MN-specific antibodies and their use in cancer treatment

ABSTRACT

A new gene--MN--and proteins/polypeptides encoded therefrom are disclosed. Recombinant nucleic acid molecules for expressing MN proteins/polypeptides and fusion proteins are provided. Expression of the MN gene is disclosed as being associated with tumorigenicity, and the invention concerns methods and compositions for detecting and/or quantitating MN antigen and/or MN-specific antibodies in vertebrate samples that are diagnostic/prognostic for neoplastic and pre-neoplastic disease. Test kits embodying the immunoassays of this invention are provided. MN-specific antibodies are disclosed that can be used diagnostically/prognostically, therapeutically, for imaging, and/or for affinity purification of MN proteins/polypeptides. Also provided are nucleic acid probes for the MN gene as well as test kits comprising said probes. The invention also concerns vaccines comprising MN proteins/polypeptides which are effective to immunize a vertebrate against neoplastic diseases associated with the expression of MN proteins. The invention still further concerns antisense nucleic acid sequences that can be used to inhibit MN gene expression.

This application is a continuation-in-part of U.S. Ser. No. 07/964,589(filed Oct. 21, 1992) which issued on Feb. 7, 1995 as U.S. Pat. No.5,387,676 and declares priority therefrom and also from now pendingCzechoslovakian patent application PV-709-92 (filed Mar. 11, 1992) under35 USC Section 119.

FIELD OF THE INVENTION

The present invention is in the general area of medical genetics and inthe fields of biochemical engineering and immunochemistry. Morespecifically, it relates to the identification of a new gene--the MNgene--a cellular gene coding for the MN protein. The inventors hereoffound MN proteins to be associated with tumorigenicity. Identificationof MN antigen as well as antibodies specific therefor in patient samplesprovides the basis for diagnostic/prognostic assays for cancer.

BACKGROUND OF THE INVENTION

MaTu is a novel quasi-viral agent with rather unusual properties[Zavada, J., Arch. Virol, 50: 1-10 (1976)]. It is presumably derivedfrom a human mammary tumor. In some respects, it resembles classicalviruses whereas in other respects, it resembles "slow" viruses (prions),and in still other respects it is different from both classes ofviruses.

MaTu was first detected by its capacity to complement mutants ofvesicular stomatitis virus (VSV) with heat-labile surface G protein inHeLa cells (cell line derived from human cervical adenocarcinoma), whichhad been cocultivated with human breast carcinoma cells. Thecomplementation resulted in the formation of phenotypically mixedvirions--the VSV(MaTu) pseudotypes [Zavada et al., Nature New Biol, 240:124-125 (1972)]. The virions contain the VSV genome, which isresponsible for their ability to produce plaques (as well as internalVSV proteins), but the surface protein, corresponding to MaTu,determines their host range and neutralization specificities.

One of the paradoxical features of the MaTu agent is its host range.VSV(MaTu) is infectious only for human fibroblasts, but not for HeLa;however, the MaTu agent, detected by its capacity to donate surfaceprotein for the VSV(MaTu) pseudotypes, is transmissible only to HeLa,but not to fibroblasts [Zavada et al., J. Gen. Virol., 24: 327-337(1974)].

By its complementation of VSV mutants and by its formation ofpseudotypes, MaTu resembles known enveloped viruses. However, MaTu istransmissible only by direct cell-to-cell contact, and not by cell-freefiltrates, thus differing from both classical and "slow" viruses. Itsonly permissive host appears to be HeLa cells. In those cells, MaTuspreads extremely slowly, and does not form morphologically distinctvirions, thus resembling the "slow" viruses. [Zavada et al., (1974);Zavada and Zavadova, Arch. Virol. 118: 189-197 (1991)]. No known virushas HeLa cells as an exclusive host.

Since the above-described properties suggest that MaTu might be anentirely new type of molecular parasite of living cells, and since itpossibly originated from a human tumor, there was a significant medicalresearch interest to characterize it in more detail. Herein elucidatedis the biological and molecular nature of MaTu. MaTu was found to be atwo-component system, having an exogenous transmissible component, MX,and an endogenous cellular component, MN. The MN gene was further foundto be present in the chromosomal DNA of all vertebrates tested, and itsexpression was found to be strongly correlated with tumorigenicity.

Described herein is the cloning and sequencing of the MN gene and theproduction of a MN-encoded protein in a bacterial vector. Thatgenetically engineered MN protein as well as other MNproteins/polypeptides, can be used in serological assays according tothis invention to detect MN-specific antibodies. Further, such MNproteins/polypeptides and antibodies reactive with MN antigen can beused in immunoassays according to this invention to detect and/orquantitate MN antigen. Such assays may be diagnostic and/or prognosticfor neoplastic and/or pre-neoplastic disease.

SUMMARY OF THE INVENTION

Herein disclosed is the MN gene, a cellular gene which is the endogenouscomponent of the MaTu agent. cDNA sequences for the MN gene are shown inFIGS. 1A-B [SEQ. ID. NO.: 1] and FIG. 15 [SEQ. ID. NO.: 5].

This invention is directed to said MN gene, fragments thereof and therelated cDNA which are useful, for example, as follows: 1) to produce MNproteins/polypeptides by biochemical engineering; 2) to prepare nucleicacid probes to test for the presence of the MN gene in cells of asubject; 3) to prepare appropriate polymerase chain reaction (PCR)primers for use, for example, in PCR-based assays or to produce nucleicacid probes; 4) to identify MN proteins and polypeptides as well ashomologs or near homologs thereto; 5) to identify various mRNAstranscribed from MN genes in various tissues and cell lines, preferablyhuman; and 6) to identify mutations in MN genes. The invention furtherconcerns purified and isolated DNA molecules comprising the MN gene orfragments thereof, or the related cDNA or fragments thereof.

Thus, this invention in one aspect concerns isolated nucleic acidsequences that encode MN proteins or polypeptides wherein the nucleotidesequences for said nucleic acids are selected from the group consistingof:

(a) SEQ. ID. NO.: 1;

(b) SEQ. ID. NO.: 5;

(c) nucleotide sequences that hybridize under stringent conditions toSEQ. ID. NO.: 1 or to its complement;

(d) nucleotide sequences that hybridize under stringent conditions toSEQ. ID. NO.: 5 or to its complement; and

(e) nucleotide sequences that differ from SEQ. ID. NO.: 1 or SEQ. IDNO.: 5, or from the nucleotide sequences of (c) and (d) in codonsequence because of the degeneracy of the genetic code, that is,sequences that are degenerate variants of those sequences. Further, suchnucleic acid sequences are selected from nucleotide sequences that butfor the degeneracy of the genetic code would hybridize to either SEQ.ID. NO.: 1 or SEQ. ID. NO.: 5 under stringent hybridization conditions.

Further, this invention concerns nucleic acid probes which are fragmentsof the isolated nucleic acids that encode MN proteins or polypeptides asdescribed above. Preferably said nucleic acid probes are comprised of atleast 50 nucleotides. Fragments of said isolated nucleic acids thatencode MN proteins or polypeptides according to this invention can, asindicated above, be used as PCR primers to amplify segments of MN genes,and may be useful in identifying mutations in MN genes.

This invention also concerns nucleic acids which encode MN proteins orpolypeptides that are specifically bound by monoclonal antibodiesdesignated M75 that are produced by the hybridoma VU-M75 deposited atthe American Type Culture Collection (ATCC) at 10801 University Blvd. inManassas, Va. 20110-2209 (USA) under ATCC No. HB 11128.

The invention further concerns the discovery of a hitherto unknownprotein--MN, encoded by the MN gene. The expresssion of MN proteins isinducible by growing cells in dense cultures, and such expression wasdiscovered to be associated with tumorigenic cells.

MN proteins were found to be produced by some human tumor cell lines invitro, for example, by HeLa (cervical carcinoma), T24 (bladdercarcinoma) and T47D (mammary carcinoma) and SK-Mel 1477 (melanoma) celllines, by tumorigenic hybrid cells and by cells of some human cancers invivo, for example, by cells of uterine cervical, ovarian and endometrialcarcinomas as well as cells of some benign neoplasias such as mammarypapillomas. MN proteins were not found in non-tumorigenic hybrid cells,and are generally not found in the cells of normal tissues, althoughthey have been found in a few normal tissues, most notably andabundantly in normal stomach tissues. MN antigen was found byimmunohistochemical staining to be prevalent in tumor cells and to bepresent sometimes in morphologically normal appearing areas of tissuespecimens exhibiting dysplasia and/or malignancy. Thus, the MN gene isstrongly correlated with tumorigenesis and is considered to be aputative oncogene.

In HeLa and in tumorigenic HeLa×fibroblast hybrid (H/F/T) cells, MNprotein is manifested as a "twin" protein p54/58N; it is glycosylatedand forms disulfide-linked oligomers. As determined by electrophoresisupon reducing gels, MN proteins have molecular weights in the range offrom about 40 kd to about 70 kd, preferably from about 45 kd to about 65kd, more preferably from about 48 kd to about 58 kd. Upon non-reducinggels, MN proteins in the form of oligomers have molecular weights in therange of from about 145 kd to about 160 kd, preferably from about 150 toabout 155 kd, still more preferably from about 152 to about 154 kd. Thepredicted amino acid sequences for preferred MN proteins of thisinvention are shown in FIGS. 1A-1B [SEQ. ID. NO. 2] and in FIGS. 15A-15C[SEQ. ID. NO.: 6].

The discovery of the MN gene and protein and thus, of substantiallycomplementary MN genes and proteins encoded thereby, led to the findingthat the expression of MN proteins was associated with tumorigenicity.That finding resulted in the creation of methods that arediagnostic/prognostic for cancer and precancerous conditions. Methodsand compositions are provided for identifying the onset and presence ofneoplastic disease by detecting and/or quantitating MN antigen inpatient samples, including tissue sections and smears, cell and tissueextracts from vertebrates, preferably mammals and more preferablyhumans. Such MN antigen may also be found in body fluids.

MN proteins and genes are of use in research concerning the molecularmechanisms of oncogenesis, in cancer diagnostics/prognostics, and may beof use in cancer immunotherapy.

The present invention is useful for detecting a wide variety ofneoplastic and/or pre-neoplastic diseases. Exemplary neoplastic diseasesinclude carcinomas, such as mammary, bladder, ovarian, uterine,cervical, endometrial, squamous cell and adenosquamous carcinomas; andhead and neck cancers; mesodermal tumors, such as neuroblastomas andretinoblastomas; sarcomas, such as osteosarcomas and Ewing's sarcoma;and melanomas. Of particular interest are head and neck cancers,gynecologic cancers including ovarian, cervical, vaginal, endometrialand vulval cancers; gastrointestinal cancer, such as, stomach, colon andesophageal cancers; urinary tract cancer, such as, bladder and kidneycancers; skin cancer; liver cancer; prostate cancer; lung cancer; andbreast cancer. Of still further particular interest are gynecologiccancers; breast cancer; urinary tract cancers, especially bladdercancer; lung cancer; gastrointestinal cancer, such as, stomach, colonand esophageal cancers; and liver cancer. Even further of particularinterest are gynecologic cancers and breast cancer. Gynecologic cancersof particular interest are carcinomas of the uterine cervix, endometriumand ovaries; more particularly such gynecologic cancers include cervicalsquamous cell carcinomas, adenosquamous carcinomas, adenocarcinomas aswell as gynecologic precancerous conditions, such as metaplasticcervical tissues and condylomas.

The invention further relates to the biochemical engineering of the MNgene, fragments thereof or related cDNA. For example, said gene or afragment thereof or related cDNA can be inserted into a suitableexpression vector; host cells can be transformed with such an expressionvector; and an MN protein/polypeptide, preferably an MN protein, isexpressed therein. Such a recombinant protein or polypeptide can beglycosylated or nonglycosylated, preferably glycosylated, and can bepurified to substantial purity. The invention further concerns MNproteins/polypeptides which are synthetically or otherwise biologicallyprepared.

Said MN proteins/polypeptides can be used in assays to detect MN antigenin patient samples and in serological assays to test for MN-specificantibodies. MN proteins/polypeptides of this invention are serologicallyactive, immunogenic and/or antigenic. They can further be used asimmunogens to produce MN-specific antibodies, polyclonal and/ormonoclonal, as well as an immune T-cell response.

The invention further is directed to MN-specific antibodies, which canbe used diagnostically/prognostically and may be used therapeutically.MN-specific antibodies can be used, for example, in laboratorydiagnostics, using immunofluorescence microscopy or immunohistochemicalstaining; as a component in immunoassays for detecting and/orquantitating MN antigen in, for example, clinical samples; as probes forimmunoblotting to detect MN antigen; in immunoelectron microscopy withcolloid gold beads for localization of MN proteins and/or polypeptidesin cells; and in genetic engineering for cloning the MN gene orfragments thereof, or related cDNA. Such MN-specific antibodies can beused as components of diagnostic/prognostic kits, for example, for invitro use on histological sections; such antibodies can also and usedfor in vivo diagnostics/prognostics, for example, such antibodies can belabeled appropriately, as with a suitable radioactive isotope, and usedin vivo to locate metastases by scintigraphy. Further such antibodiesmay be used in vivo therapeutically to treat cancer patients with orwithout toxic and/or cytostatic agents attached thereto. Further, suchantibodies can be used in vivo to detect the presence of neoplasticand/or pre-neoplastic disease. Still further, such antibodies can beused to affinity purify MN proteins and polypeptides.

A hybridoma that produces a representative MN-specific antibody, themonoclonal antibody M75, was deposited at the American Type CultureCollection [ATCC; 10801 University Blvd. in Manassas, Va. 20110-2209(USA)] on Sep. 17, 1992, under ATCC Number HB 11128. The M75 antibodywas used to discover and identify the MN protein and can be used toreadily identify MN antigen in Western blots, in radioimmunoassays andimmunohistochemically, for example, in tissue samples that are fresh,frozen, or formalin-, alcohol-, acetone- or otherwised fixed and/orparaffin-embedded and deparaffinized.

This invention also concerns recombinant DNA molecules comprising a DNAsequence that encodes for an MN protein or polypeptide, and alsorecombinant DNA molecules that encode not only for an MN protein orpolypeptide but also for an amino acid sequence of a non-MN protein orpolypeptide. Said non-MN protein or polypeptide may preferably benonimmunogenic to humans and not typically reactive to antibodies inhuman body fluids. Examples of such a DNA sequence is the alpha-peptidecoding region of beta-galactosidase and a sequence coding forglutathione S-transferase or a fragment thereof. However, in someinstances, a non-MN protein or polypeptide that is serologically active,immunogenic and/or antigenic may be preferred as a fusion partner to aMN antigen. Further, claimed herein are such recombinant fusionproteins/polypeptides which are substantially pure and non-naturallyoccurring. An exemplary fusion protein of this invention is pGEX-3X-MN.

This invention also concerns methods of treating neoplastic diseaseand/or pre-neoplastic disease comprising inhibiting the expression of MNgenes by administering antisense nucleic acid sequences that aresubstantially complementary to mRNA transcribed from MN genes. Saidantisense nucleic acid sequences are those that hybridize to such mRNAunder stringent hybridization conditions. Preferred are antisensenucleic acid sequences that are substantially complementary to sequencesat the 5' end of the MN cDNA sequence shown in FIGS. 1A-1B. Preferablysaid antisense nucleic acid sequences are oligonucleotides.

This invention also concerns vaccines comprising an immunogenic amountof one or more substantially pure MN proteins and/or polypeptidesdispersed in a physiologically acceptable, nontoxic vehicle, whichamount is effective to immunize a vertebrate, preferably a mammal, morepreferably a human, against a neoplastic disease associated with theexpression of MN proteins. Said proteins can be recombinantly,synthetically or otherwise biologically produced. Recombinent MNproteins includes fusion proteins, as exemplified by pGEX-3X-MN. Aparticular use of said vaccine would be to prevent recidivism and/ormetastasis. For example, it could be administered to a patient who hashad an MN-carrying tumor surgically removed, to prevent recurrence ofthe tumor.

The invention still further concerns nucleic acid probes, preferablycontaining at least 50 nucleotides, that are substantially complementaryto nucleic acid sequences of the MN gene. Preferred nucleic acid probesof this invention are those with sequences substantially complementaryto the sequences from MN cDNA shown in FIGS. 1A-1B [SEQ. ID. NO.: 1] and15 [SEQ. ID. NO.: 5]. Preferred nucleic acid probes of this inventionare fragments of the isolated nucleic acids that encode MN proteins andpolypeptides, which fragments contain at least 50 nucleotides, and whichhybridize under stringent conditions to either SEQ. ID. NOS.: 1 or 5 orto the complementary strands to SEQ. ID. NOS. 1 or 5. Test kits of thisinvention can comprise such probes which are usefuldiagnostically/prognostically for neoplastic and/or pre-neoplasticdisease. Preferred test kits comprise means for detecting or measuringthe hybridization of said probes to the MN gene or to the mRNA productof the MN gene, such as a visualizing means.

The immunoassays of this invention can be embodied in test kits whichcomprise MN proteins/polypeptides and/or MN-specific antibodies. Suchtest kits can be in solid phase formats, but are not limited thereto,and can also be in liquid phase format, and can be based onimmunohistochemical assays, ELISAS, particle assays, radiometric orfluorometric assays either unamplified or amplified, using, for example,avidin/biotin technology.

Abbreviations

The following abbreviations are used herein:

AA--amino acid

ATCC--American Type Culture Collection

bp--base pairs

BSA--bovine serum albumin

CA--carbonic anhydrase

Ci--curie

cm--centimeter

cpm--counts per minute

C-terminus--carboxyl-terminus

° C.--degrees centigrade

DMEM--Dulbecco modified Eagle medium

EDTA--ethylenediaminetetracetate

EIA--enzyme immunoassay

ELISA--enzyme-linked immunosorbent assay

F--fibroblasts

FCS--fetal calf serum

FIBR--fibroblasts

FITC--fluorescein isothiocyanate

H--HeLa cells

HEF--human embryo fibroblasts

HeLa K--standard type of HeLa cells

HeLa S--Stanbridge's mutant HeLa D98/AH.2

H/F-T--hybrid HeLa fibroblast cells that are tumorigenic; derived fromHeLa D98/AH.2

H/F-N--hybrid HeLa fibroblast cells that are nontumorigenic; derivedfrom HeLa D98/AH.2

HGPRT⁻ --hypoxanthine guanine phosphoribosyl transferase-deficient

HRP--horseradish peroxidase

IPTG--isopropyl-Beta-D-thiogalacto-pyranoside

kb--kilobase

kd--kilodaltons

M--molar

mA--milliampere

MAb--monoclonal antibody

ME--mercaptoethanol

MEM--minimal essential medium

mg--milligram

ml--milliliter

mM--millimolar

MTV--mammary tumor virus

N--normal concentration

ng--nanogram

N-terminus--amino-terminus

ODN--oligodeoxynucleotide

PAGE--polyacrylamide gel electrophoresis

PBS--phosphate buffered saline

PEST--combination of one-letter abbreviations for proline, glutamicacid, serine, threonine

pI--isoelectric point

RIP--radioimmunoprecipitation

RIPA--radioimmunoprecipitation assay

SAC--protein A-Staphylococcus aureus cells

SDS--sodium dodecyl sulfate

SDS-PAGE--sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SSPE--NaCl (0.18 M), sodium phosphate (0.01 M), EDTA (0.001 M)

TCA--trichloroacetic acid

TC--media tissue culture media

Tris--tris (hydroxymethyl) aminomethane

μCi--microcurie

μg--microgram

μl--microliter

μM--micromolar

VSV--vesicular stomatitis virus

X-MLV--xenotropic murine leukemia virus

Cell Lines

The following cell lines were used in the experiments herein described:

HeLa K--standard type of HeLa cells; aneuploid, epithelial-like cellline isolated from a human cervical adenocarcinoma [Gey et al., CancerRes., 12: 264 (1952); Jones et al., Obstet. Gynecol., 38: 945-949(1971)] obtained from Professor B. Korych, [Institute of MedicalMicrobiology and Immunology, Charles University; Prague, Czech Republic]

HeLa D98/AH.2--Mutant HeLa clone that is hypoxanthine (also HeLa S)guanine phosphoribosyl transferase-deficient (HGPRT⁻) kindly provided byEric J. Stanbridge [Department of Microbiology, College of Medicine,University of California, Irvine, Calif. (USA)] and reported inStanbridge et al., Science, 215: 252-259 (Jan. 15. 1982); parent ofhybrid cells H/F-N and H/F-T, also obtained from E. J. Stanbridge.

NIH-373--murine fibroblast cell line reported in Aaronson, Science, 237:178 (1987).

T47D--cell line derived from a human mammary carcinoma [Keydar et al.,Eur. J. Cancer, 15: 659-670 (1979)]; kindly provided by J. Keydar[Haddasah Medical School; Jerusalem, Israel]

T24--cell line from urinary bladder carcinoma [Bubenik et al., Int. J.Cancer, 11: 765-773 (1973)] kindly provided by J. Bubenik [Institute ofMolecular Genetics, Czechoslovak Academy of Sciences; Prague, CzechRepublic]

HMB2--cell line from melanoma [Svec et al., Neoplasma, 35: 665-681(1988)]

HEF--human embryo fibroblasts [Zavada et al., Nature New Biology, 240:124-125 (1972)]

SIRC--cell line from rabbit cornea (control and X-MLV-infected) [Zavadaet al., Virology, 82: 221-231 (1977)]

Vero cells--African green monkey cell line [Zavada et al. (1977)]

myeloma cell line NS-0--myeloma cell line used as a fusion parent inproduction of monoclonal antibodies [Galfre and Milstein, MethodsEnzymol., 73: 3-46 (1981)]

SK-Mel 1477--human melanoma cell line kindly provided by K. E. Hellstrom[Division of Tumor Immunology, Fred Hutchins Cancer Research Center;Seattle, Wash. (USA)]

XC--cells derived from a rat rhabdomyosarcoma induced with Rous sarcomavirus-induced rat sarcoma [Svoboda, J., Natl. Cancer Center InstituteMonograph No. 17, IN: "International Conference on Avian Tumor Viruses"(J. W. Beard ed.), pp. 277-298 (1964)], kindly provided by Jan Svoboda[Institute of Molecular Genetics, Czechoslovak Academy of Sciences;Prague, Czech Republic]; and

Rat 2-Tk⁻ --a thymidine kinase deficient cell line, kindly provided byL. Kutinova [Institute of Sera and Vaccines; Prague, Czech Republic]

CGL1--H/F-N hybrid cells (HeLa D98/AH.2 derivative)

CGL2--H/F-T hybrid cells (HeLa D98/AH.2 derivative)

CGL3--H/F-T hybrid cells (HeLa D98/AH.2 derivative)

CGL4--H/F-T hybrid cells (HeLa D98/Ah.2 derivative)

Nucleotide and Amino Acid Sequence Symbols

The following symbols are used to represent nucleotides herein:

    ______________________________________                                        Base Symbol        Meaning                                                    ______________________________________                                        A                  adenine                                                    C                  cytosine                                                   G                  guanine                                                    T                  thymine                                                    U                  uracil                                                     I                  inosine                                                    M                  A or C                                                     R                  A or G                                                     W                  A or T/U                                                   S                  C or G                                                     Y                  C or T/U                                                   K                  G or T/U                                                   V                  A or C or G                                                H                  A or C or T/U                                              D                  A or G or T/U                                              B                  C or G or T/U                                              N/X                A or C or G or T/U                                         ______________________________________                                    

There are twenty main amino acids, each of which is specified by adifferent arrangement of three adjacent nucleotides (triplet code orcodon), and which are linked together in a specific order to form acharacteristic protein. A three-letter or one-letter convention is usedherein to identify said amino acids, as, for example, in FIGS. 1A-B andFIGS. 15A-15C, respectively, as follows:

    ______________________________________                                        Amino acid name                                                                              3 Ltr. Abbrev.                                                                           1 Ltr. Abbrev.                                      ______________________________________                                        Alanine        Ala        A                                                   Arginine       Arg        R                                                   Asparagine     Asn        N                                                   Aspartic Acid  Asp        D                                                   Cysteine       Cys        C                                                   Glutamic Acid  Glu        E                                                   Glutamine      Gln        Q                                                   Glycine        Gly        G                                                   Histidine      His        H                                                   Isoleucine     Ile        I                                                   Leucine        Leu        L                                                   Lysine         Lys        K                                                   Methionine     Met        M                                                   Phenylalanine  Phe        F                                                   Proline        Pro        P                                                   Serine         Ser        S                                                   Threonine      Thr        T                                                   Tryptophan     Trp        W                                                   Tyrosine       Tyr        Y                                                   Valine         Val        V                                                   ______________________________________                                    

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B provides the nucleotide sequence for a MN cDNA [SEQ. ID.NO.: 1] clone isolated as described herein and the predicted amino acidsequence [SEQ. ID. NO.: 2] encoded by the cDNA. That sequence data hasbeen sent to the EMBL Data Library in Heidelberg, Germany and isavailable under Accession No. X66839.

FIG. 2 provides SDS-PAGE and immunoblotting analyses of recombinant MNprotein expressed from a pGEX-3X bacterial expression vector. Twoparallel samples of purified recombinant MN protein (twenty μg in eachsample) were separated by SDS-PAGE on a 10% gel. One sample (A in FIG.2) was stained with Coomassie brilliant blue; whereas the other sample(B) was blotted onto a Hybond C membrane [Amersham; Aylesbury, Bucks,England]. The blot was developed by autoradiography with ¹²⁵ I-labeledMab M75.

FIG. 3 illustrates inhibition of p54/58 expression by antisenseoligodeoxynucleotides (ODNs). HeLa cells cultured in overcrowdedconditions were incubated with (A) 29-mer ODNI [SEQ. ID. NO.: 3]; (B)19-mer ODN2 [SEQ. ID. NO.: 4]; (C) both ODNI and ODN2; and (D) withoutODNS. Example 11 provides details of the procedures used.

FIG. 4 shows the results of Northern blotting of MN mRNA in human celllines. Total RNA was prepared from the following cell lines: HeLa cellsgrowing in dense (A) and sparse (B) culture; (C) H/F-N; (D) and (E)H/F-T; and (F) human embryo fibroblasts. Example 12 details theprocedure and results.

FIG. 5 illustrates the detection of the MN gene in genomic DNAs bySouthern blotting. Chromosomal DNA digested by PstI was as follows: (A)chicken; (B) bat; (C) rat; (D) mouse; (E) feline; (F) pig; (G) sheep;(H) bovine; (I) monkey; and (J) human HeLa cells. The procedures usedare detailed in Example 13.

FIGS. 6A-6B graphically illustrates the expression of MN- andMX-specific proteins in human fibroblasts (F), in HeLa cells (H) and inH/F-N and H/F-T hybrid cells and contrasts the expression in MX-infectedand MX-uninfected cells. Example 5 details the procedures and results.

FIG. 7 (discussed in Example 5) provides immunoblots of MN proteins infibroblasts (FIBR) and in HeLa K, HeLa S, H/F-N and H/F-T hybrid cells.

FIG. 8 (discussed in Example 6) shows immunoblots of MN proteins in cellculture extracts prepared from the following: (A) MX-infected HeLacells; (B) human fibroblasts; (C) T24; (D) T47D; (E) SK-Mel 1477; and(F) HeLa cells uninfected with MX. The symbols +ME and O ME indicatethat the proteins were separated by PAGE after heating in a samplebuffer, with and without 3% mercaptoethanol (ME), respectively.

FIG. 9 (discussed in Example 6) provides immunoblots of MN proteins fromhuman tissue extracts. The extracts were prepared from the following:(A) MX-infected HeLa cells; (B) full-term placenta; (C) corpus uteri;(D, M) adenocarcinoma endometrii; (E, N) carcinoma ovarii; (F, G)trophoblasts; (H) normal ovary; (I) myoma uteri; (J) mammary papilloma;(K) normal mammary gland; (L) hyperplastic endometrium; (O) cervicalcarcinoma; and (P) melanoma. FIG. 10 (discussed in Example 7) providesimmunoblots of MN proteins from (A) MX-infected HeLa cells and from (B)Rat2-Tk⁻ cells. (+ME and 0 ME have the same meanings as explained in thelegend to FIG. 8.)

FIGS. 11A-11B (discussed in Example 8) graphically illustrates theresults from radioimmunoprecipitation experiments with ¹²⁵ I-pGEX-3X-MNprotein and different antibodies. The radioactive protein (15×10³cpm/tube) was precipitated with ascitic fluid or sera and SAC asfollows: (A) ascites with MAb M75; (B) rabbit anti-MaTu serum; (C)normal rabbit serum; (D) human serum L8; (E) human serum KH; and (F)human serum M7.

FIG. 12 (discussed in Example 8) shows the results fromradioimmunoassays for MN antigen. Ascitic fluid (dilution precipitating50% radioactivity) was allowed to react for 2 hours with (A) "cold"(unlabeled) protein pGEX-3X-MN, or with extracts from cells as follows:(B) HeLa+MX; (C) Rat-2Tk⁻ ; (D) HeLa; (E) rat XC; (F) T24; and (G) HEF.Subsequently ¹²⁵ I-labeled pGEX-3X-MN protein (25×10³ cpm/tube) wasadded and incubated for an additional 2 hours. Finally, theradioactivity to MAb M75 was adsorbed to SAC and measured.

FIGS. 13(A-F) (discussed in Example 10) provide results ofimmunoelectron and scanning microscopy of MX-uninfected (control) andMX-infected HeLa cells. Panels A-D show ultrathin sections of cellsstained with MAb M75 and immunogold; Panels E and F are scanningelectron micrographs of cells wherein no immunogold was used. Panels Eand F both show a terminal phase of cell division. Panels A and E are ofcontrol HeLa cells; panels B, C, D and F are of MX-infected HeLa cells.The cells shown in Panels A, B and C were fixed and treated with M75 andimmunogold before they were embedded and sectioned. Such a procedureallows for immunogold decoration only of cell surface antigens. Thecells in Panel D were treated with M75 and immunogold only once they hadbeen embedded and sectioned, and thus antigens inside the cells couldalso be decorated.

FIG. 14 compares the results of immunizing baby rats to XC tumor cellswith rat serum prepared against the fusion protein MN glutathioneS-transferase (pGEX-3X-MN) (the IM group) with the results of immunizingbaby rats with control rat sera (the C group). Each point on the graphrepresents the tumor weight of a tumor from one rat. Example 15 detailsthose experiments.

FIGS. 15A-C shows a complete nucleotide sequence of a MN cDNA [SEQ. ID.NO.: 5]. Also shown is the deduced amino acid sequence [SEQ. ID. NO.:6]. The polyadenylation signal (AATAAA) and the mRNA instability motif(ATTTA) are located at nucleotides (nts) 1507-1512 and at nts 1513-1518,respectively. The amino acid residues of the putative signal peptide aswell as the membrane-spanning segment are located at amino acids (aa)1-37 and at aa 415-433, respectively. The N-glycosylation site islocated at aa 346. The S/TPXX elements are located at amino acids 7-10,47-50, 71-74, 153-156, 162-165, 333-336, and 397-400. The cDNA sequenceof FIGS. 15A-15C has been entered into the EMBL Data Library under theaccession number X66839.

FIG. 16 is a restriction map of the full-length MN cDNA. The openreading frame is shown as an open box. The thick lines below therestriction map illustrate the sizes and positions of two overlappingcDNA clones. The horizontal arrows indicate the positions of primers R1[SEQ. ID. NO.: 7] and R2 [SEQ. ID. NO.: 8] used for the 5' end RACE.Relevant restriction sites are BamHI (B), EcoRV (V), EcoRI (E), PstI(Ps), PvuII (Pv).

FIG. 17 shows a restriction analysis of the MN gene. Genomic DNA fromHeLa cells was cleaved (as described in Example 13) with the followingrestriction enzymes: EcoRI (1), EcoRV (2), HindIII (3), KpnI (4), NcoI(5), PstI (6), and PvuII (7), and then analyzed by Southernhybridization under stringent conditions using MN cDNA as a probe.

DETAILED DESCRIPTION

MaTu--MX and MN Components

As demonstrated herein MaTu is a two-component system. One part of thecomplex, exogenous MX, is transmissible, and is manifested by a protein,p58X, which is a cytoplasmic antigen which reacts with some naturalsera, of humans and of various animals. The other component, MN, isendogenous to human cells. The level of MN expression has been found tobe considerably increased in the presence of the MaTu-MX transmissibleagent, which has been recently identified as lymphocyticchoriomeningitis virus (LCMV) which persistently infects HeLa cells.

MN is a cellular gene, showing only very little homology with known DNAsequences. It is rather conservative and is present as a single copygene in the chromosomal DNA of various vertebrates. Described herein isthe cloning and sequencing of the MN cDNA, and the genetic engineeringof a fusion protein, namely MN plus the carboxyl terminus of glutathioneS-transferase, that can be conveniently purified by affinitychromatography.

MN is manifested in HeLa cells by a twin protein, p54/58N, that islocalized on the cell surface and in the nucleus. Immunoblots using amonoclonal antibody reactive with p54/58N (MAb M75) revealed two bandsat 54 kd and 58 kd. Those two bands may correspond to one type ofprotein that differs by glycosylation pattern or by how it is processed.(Both p54N and p58N are glycosylated with oligosaccharidic residuescontaining mannose, but only p58N also contains glucosamine.) Herein,the phrase "twin protein" indicates p54/58N.

MN is absent in rapidly growing, sparse cultures of HeLa, but isinducible either by keeping the cells in dense cultures or, moreefficiently, by infecting them with MX (LCMV). Those inducing factorsare synergistic. only p54/58N is associated with virions of vesicularstomatitis virus (VSV), reproduced in MaTu-infected HeLa. Whereas thetwin protein p54/58N is glycosylated and forms oligomers linked bydisulfidic bonds, p58X is not glycosylated and does not form S--S-linkedoligomers.

VSV assembles p54/58N into virions in HeLa cells, indicating that thetwin protein is responsible for complementation of VSV G-protein mutantsand for formation of VSV(MaTu) pseudotypes. As only enveloped virusesprovide surface glycoproteins for the formation of infectious,functioning pseudotypes, which can perform such specific functions asadsorption and penetration of virions into cells [Zavada, J., J. Gen.Virol., 63: 15-24 (1982)], that observation implies that the MN genebehaves as a quasi-viral sequence.

The surface proteins of enveloped viruses, which participate in theformation of VSV pseudotypes, are glycosylated as is the MN twinprotein, p54/58N. MN proteins also resemble viral glycoproteins in theformation of oligomers (preferably tri- or tetramers); sucholigomerization, although not necessarily involving S--S bonds(disulfidic bonds), is essential for the assembly of virions [Kreis andLodish, Cell, 46: 929-937 (1986)]. The disulfidic bonds can be disruptedby reduction with 2-mercaptoethanol.

As reported in Pastorekova et al., Virology, 187: 620-626 (1992), afterreduction with mercaptoethanol, p54/58N from cell extracts or from VSVlooks very similar on immunoblot. Without reduction, in cell extracts,it gives several bands around 150 kd, suggesting that the cells maycontain several different oligomers (probably with a different p54:p58ratio), but VSV selectively assembles only one of them, with a molecularweight of about 153 kd. That oligomer might be a trimer, or rather atetramer, consisting of 54 kd and 58 kd proteins. The equimolar ratio ofp54:p58 in vsv virions is indicated by approximately the same strengthof 54 kd and 58 kd bands in a VSV sample analyzed under reducingconditions.

The expression of MN proteins appears to be diagnostic/prognostic forneoplastic disease. The MN twin protein, p54/58N, was found to beexpressed in HeLa cells and in Stanbridge's tumorigenic (H/F-T) hybridcells [Stanbridge et al., Somatic Cell Genet, 7: 699-712 (1981); andStanbridge et al., Science, 215: 252-259 (1982)] but not in fibroblastsor in non-tumorigenic (H/F-N) hybrid cells [Stanbridge et al., id.]. Inearly studies, MN proteins were found in immunoblots prepared from humanovarian, endometrial and uterine cervical carcinomas, and in some benignneoplasias (as mammary papilloma) but not from normal ovarian,endometrial, uterine or placental tissues. Example 14 herein detailsfurther research on MN gene expression wherein MN antigen, as detectedby immunohistochemical staining, was found to be prevalent in tumorcells of a number of cancers, including cervical, bladder, head andneck, and renal cell carcinomas among others. Further, theimmunohistochemical staining experiments of Example 14 show that amongnormal tissues tested, only normal stomach tissues showed routinely andextensively the presence of MN antigen. MN antigen is further shownherein to be present sometimes in morphologically normal-appearing areasof tissue specimens exhibiting dysplasia and/or malignancy.

In HeLa cells infected with MX, observed were conspicuousultrastructural alterations, that is, the formation of abundantfilaments on cell surfaces and the amplification of mitochondria. Usingan immunogold technique, p54/58N was visualized on the surface filamentsand in the nucleus, particularly in the nucleoli. Thus MN proteinsappear to be strongly correlated with tumorigenicity, and do not appearto be produced in general by normal non-tumor cells.

The examples herein show that MX and MN are two different entities, thatcan exist independently of each other. MX (LCMV) as an exogenous,transmissible agent can multiply in fibroblasts and in H/F-N hybridcells which are not expressing MN-related proteins (FIGS. 6A-6B). Insuch cells, MX does not induce the production of MN protein. MN proteincan be produced in HeLa and other tumor cells even in the absence of MXas shown in FIGS. 6A-6B through 9. However, MX is a potent inducer ofMN-related protein in HeLa cells; it increases its production thirtytimes over the concentration observed in uninfected cells (FIGS. 7 and12, Table 1 in Example 8, below).

MN Gene--Cloning and Sequencing and Deduced Amino Acid Sequences

FIGS. 1A-1B provides the nucleotide sequence for a MN cDNA cloneisolated as described within this section. FIGS. 15A-15C provides thecomplete nucleotide sequence for the MN cDNA.

It is understood that because of the degeneracy of the genetic code,that is, that more than one codon will code for one amino acid [forexample, the codons TTA, TTG, CTT, CTC, CTA and CTG each code for theamino acid leucine (leu)], that variations of the nucleotide sequencesin, for example, the sequences shown in FIGS. 1A-B and in FIGS. 15A-15C,wherein one codon is substituted for another, would produce asubstantially equivalent protein or polypeptide according to thisinvention. All such variations in the nucleotide sequences of the MNcDNA and complementary nucleic acid sequences are included within thescope of this invention.

It is further understood that the nucleotide sequences herein describedand shown in FIGS. 1A-1B and 15A-15C represent only the precisestructures of the cDNA nucleotide sequences isolated and describedherein. It is expected that slightly modified nucleotide sequences willbe found or can be modified by techniques known in the art to code forsubstantially similar MN proteins and polypeptides, for example, thosehaving similar epitopes, and such nucleotide sequences andproteins/polypeptides are considered to be equivalents for the purposeof this invention. DNA or RNA having equivalent codons is consideredwithin the scope of the invention, as are synthetic nucleic acidsequences that encode proteins/polypeptides homologous or substantiallyhomologous to MN proteins/polypeptides, as well as those nucleic acidsequences that would hybridize to said sequences under stringentconditions or that but for the degeneracy of the genetic code wouldhybridize to said cDNA nucleotide sequences under stringenthybridization. Modifications and variations of nucleic acid sequences asindicated herein are considered to result in sequences that aresubstantially the same as the MN sequence and fragments thereof.

To find the MN gene, a lambda gt11 cDNA library from MX-infected HeLacells was prepared. Total RNA from MX-infected HeLa cells was isolatedby a guanidinium-thiocyanate-CsCl method, and the mRNA was affinityseparated on oligo dT-cellulose. The synthesis of the cDNA and itscloning into lambda gt11 was carried out using kits from Amersham,except that the EcoRI-NotI adaptor was from Stratagene [La Jolla, Calif.(USA)]. The library was subjected to immunoscreening with monoclonalantibody M75 in combination with goat anti-mouse antibodies conjugatedwith alkaline phosphatase. That immunoscreening method is described inYoung and Davis, PNAS (USA), 80: 1194-1198 (1983). One positive clonewas picked from 350,000 screened plaques (representing about one-half ofthe whole library).

The positive clone was subcloned into the NotI site of pBluescript KS[Stratagene] thereby creating pBluescript-MN. Two oppositely orientednested deletions were made using Erase-a-Base™ kit [Promega; Madison,Wis. (USA)] and sequenced by dideoxy method with a T7 sequencing kit[Pharmacia; Piscataway, N.J. (USA)]. The sequencing showed a partialcDNA clone, the insert being 1397 bp long. That sequence is shown inFIGS. 1A-1B. The sequence comprises a large 1290 bp open reading frameand 107 bp 3' untranslated region containing a polyadenylation signal(AATAAA). Another interesting feature of the sequence is the presence ofa region contributing to instability of the mRNA (AUUUA at position1389) which is characteristic for mRNAs of some oncogenes andlymphokines [Shaw and Kamen, Cell, 46: 659-667 (1986)]. However, thesequence surrounding the first ATG codon in the open reading frame (ORF)did not fit the definition of a translational start site. In addition,as follows from a comparison of the size of the MN clone with that ofthe corresponding mRNA in a Northern blot (FIG. 4), the cDNA was missingabout 100 bp from the 5' end of its sequence.

Attempts to isolate a full-length clone from the original cDNA libraryfailed. Therefore, we performed a rapid amplification of cDNA ends(RACE) using MN-specific primers, R1 and R2, derived from the 5' regionof the original cDNA clone. The RACE product was inserted intopBluescript, and the entire population of recombinant plasmids wassequenced with an MN-specific primer ODN1. In that way, we obtained areliable sequence at the very 5' end of the MN cDNA as shown in FIGS.15A-15C [SEQ. ID. NO.: 5].

Specifically, RACE was performed using 5' RACE System (GIBCO BRL) asfollows. 1 μg of mRNA (the same as above) was used as a template for thefirst strand cDNA synthesis which was primed by the MN-specificantisense oligonucleotide, R1 (5'-TGGGGTTCTTGAGGATCTCCAGGAG-3') [SEQ.ID. NO.: 7]. The first strand product was precipitated twice in thepresence of ammonium acetate and a homopolymeric C tail was attached toits 3' end by TdT. Tailed cDNA was then amplified by PCR using a nestedprimer, R2 (5'-CTCTAACTTCAGGGAGCCCTCTTCTT-3') [SEQ. ID. NO.: 8] and ananchor primer that anneals to the homopolymeric tail(5'-CUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3') [SEQ. ID. NO.:9]. Amplified product was digested with BamHI and SalI restrictionenzymes and cloned into pBluescript II KS plasmid. After transformation,plasmid DNA was purified from the whole population of transformed cellsand used as a template for the sequencing with the MN-specific primerODN1 [SEQ. ID. NO.: 3; a 29-mer, the sequence for which is shown inExample 11].

The full-length MN cDNA sequence is 1519 base pairs (bp) long (FIGS.15A-15C). It contains a single ORF of 1400 bp, starting at position 12,with an ATG codon that is in a good context (GCGCATGG) with the ruleproposed for translation initiation [Kozak, J. Cell. Biol., 108: 229-241(1989)]. The AT rich 3' untranslated region contains, as indicatedabove, a polyadenylation signal (AATAAA) preceding the end of the cDNAby 10 bp. Surprisingly, the sequence from the original clone as well asfrom four additional clones obtained from the same cDNA library did notreveal any poly(A) tail. Moreover, also as indicated above, justdownstream of the poly(A) signal we found an ATTTA motif that is thoughtto contribute to mRNA instability (Shaw and Kamen, supra). This factraised possibility that the poly (A) tail is missing due to the specificdegradation of the MN mRNA.

The open reading frame of the MN cDNA clone shown in FIGS. 1A-1B encodesa putative protein of about 48 kd. Analysis of the deduced translatedamino acid (AA) sequence failed to show any significant homology topublished protein sequences. The closest homology found was that of theC-terminal part of the MN protein and different types of carbonicanhydrase (about 30-35% in 170-200 AA overlap). The active site as wellas the Zn²⁺ binding domain of carbonic anhydrase are well-conserved inthe MN protein. However, the MN gene is clearly a novel sequence derivedfrom the human genome.

The overall sequence homology between the cDNA MN sequence and cDNAsequences encoding different carbonic anhydrase (CA) isoenzymes is inthe range of 48-50%. That homology range is considered by ones in theart to be low, and therefore, the MN cDNA sequence is not closelyrelated to any CA cDNA sequences. Only very closely related sequenceshaving a homology of at least 80-90% would hybridize to each other understringent conditions. A sequence comparison of the MN cDNA sequenceshown in FIGS. 1A-1B and a corresponding cDNA of the human carbonicanhydrase II (CA II) showed that there are no stretches of identitybetween the two sequences that would be long enough to allow for asegment of the CA II cDNA sequence having 50 or more nucleotides tohybridize under stringent hybridization conditions to the MN cDNA orvice versa.

Although as indicated, the MN gene shows some homology with knowncarbonic anhydrases, it differs from them in several repects. Sevencarbonic anhydrases are known [Dodgson et al. (eds.), The CarbonicAnhydrases, (Plenum Press; New York/London (1991)]. Each of their genescontains seven introns. Also, all the known carbonic anhydrases areproteins of about 30 kd, smaller than the p54/58N-related products ofthe MN gene. Further, the carbonic anhydrases do not form oligomers asdo the MN-related proteins.

From the predicted amino acid sequence shown in FIGS. 1A-1B [SEQ. ID.NO.: 1], it is evident that the product of the MN gene is a basicprotein (pI 9.08), with one potential N-glycosylation site located atthe amino acid positions 303-313. Those observations correspond to thefinding that p54/58N proteins from HeLa cells are sensitive to Endo Hand Endo F cleavage, which causes a loss of about 3 kd each. Thehydrophilicity profile reveals a hydrophobic sequence of amino acids (atpositions 371-395) probably representing the region spanning the plasmamembrane and containing also a potential cleavage signal. The profilefits well with the observation that p54/58N proteins are localized onthe cell membrane. There are no PEST regions in the MN amino acidsequence, suggesting that the product of the MN gene is a stablelong-lived protein [Rogers et al., Science, 234: 364-368 (1986)]. Such afeature explains our experience with inefficient metabolic labeling ofp54/58N. The deduced amino acid sequence displays also other featuresnamely, 10 potential phosphorylation and 7 myristylation sites, and 3antigenic determinants.

The ORF of the MN cDNA shown in FIGS. 15A-15C has the coding capacityfor a 466 amino acid protein with a calculated molecular weight of 51.5kd. As assessed by amino acid sequence analysis, the deduced primarystructure of the MN protein can be divided into four distinct regions.The initial hydrophobic region of 37 amino acids (AA) corresponds to asignal peptide. The mature protein has an N-terminal part of 377 AA, ahydrophobic transmembrane segment of 20 AA and a C-terminal region of 32AA. The overall amino acid composition is rather basic, with a predictedisoelectric point of 9.12. The MN protein is rich in leucine (11.16%),proline (10.3%), alanine (9.44%), arginine (9.23%), and serine (9.01%).

More detailed insight into MN protein primary structure disclosed thepresence of several consensus sequences. One potential N-glycosylationsite was found at position 345 of FIGS. 15A-15C, and a putative nuclearlocalization signal composed of a stretch of basic amino acids RRARKK[Blank et al., EMBO J., 10: 4159-4167 (1991); Wang and Reed, Nature,364: 121-126 (1993)] was recognized in the middle of the protein, atposition 279-284. These features, together with the predictedmembrane-spanning region mentioned above, are consistent with theresults, in which MN was shown to be an N-glycosylated protein localizedboth in the plasma membrane and in the nucleus [Pastorekova et al.,Virol., 187: 620-626 (1992), Zavada et al., Int. J. Cancer, 54: 268-274(1993)]. MN protein sequence deduced from cDNA was also found to containsix S/TPXX sequence elements (one of them is in the signal peptide)defined by Suzuki, J. Mol. Biol., 207: 61-84 (1989) as motifs frequentlyfound in gene regulatory proteins. However, only two of them arecomposed of the suggested consensus amino acids.

The possibility that the 4 kd difference between the molecular weightsof the two MN proteins is caused by different glycosylation was ruledout, since, as indicated above, after in vitro treatment withendoglycosidases H and F, respectively, both peptide portions lost about3 kd in weight. This result indicates, in addition, that the molecularweight of the smaller 54 kd MN protein without its 3 kd sugar moiety,roughly corresponds to the molecular weight of MN calculated from thecDNA. Western blot analysis of MN proteins from cervical carcinoma andnormal stomach shows that in both tissues MN protein consists of two 54and 58 kd peptide portions.

To determine whether both p54/58N proteins were encoded by one gene,antisense ODNs were used to inhibit specifically MN gene expression.[Such use of antisense ODNs is reviewed in Stein and Cohen, Cancer Res.,48: 2659-2668 (1988).] Those experiments are detailed in Example 11. Thefindings indicated that cultivation of HeLa cells with ODNs resulted ina considerable inhibition of p54/58N synthesis, whereas the amount ofdifferent HeLa cell proteins produced remained approximately the same.Further, and importantly on immunoblotting, the specific inhibition byODNs affected both of the p54/58N proteins (FIG. 3). Thus, it wasconcluded that the MN gene that was cloned codes for both of the p54/58Nproteins in HeLa cells.

To confirm whether the gene that was cloned codes for thep54/58N-specific protein, it was subcloned into the bacterial expressionvector pGEX-3X [Pharmacia; Upsala, Sweden], constructed to express afusion protein containing the C-terminus of glutathione S-transferase.That subcloning is representative of one method to genetically engineeran MN-related protein of this invention. The following description isexemplary and not meant to limit the invention in any way.

Production of Fusion Protein pGEX-3X-MN

The cDNA insert from the above-described pBluescript-MN was released bydigesting the plasmid DNA by NotI. It was then treated with S1 nucleaseto obtain blunt ends and then cloned into a dephosphorylated SmaI siteof pGEX-3X (Pharmacia). After transformation of XL1-Blue cells andinduction with IPTG, a fusion protein was obtained.

The fusion protein--MN glutathione S-transferase was purified byaffinity chromatography on Glutathione-S-Sepharose 4B (Pharmacia).Twenty micrograms of the purified recombinant protein in each of twoparallel samples were separated by SDS-PAGE on a 10% gel. One of thesamples (A) was stained with Coomassie brilliant blue, whereas the other(B) was blotted onto a Hybond C membrane (Amersham). The blot wasdeveloped by autoradiography with ¹²⁵ I-labeled MAb M75. The results areshown in FIG. 2.

SDS-PAGE analysis provided an interesting result: a number of proteinbands with different molecular weights (FIG. 2A). A similar SDS-PAGEpattern was obtained with another representative fusion protein producedaccording to this invention, beta-galactosidase-MN that was expressedfrom lambda gt11 lysogens. It appears that those patterns are due totranslation errors caused by the presence of 9 AGGAGG codon tandems inthe MN sequence. The use of those codons is strongly avoided inbacterial genes because of the shortage of corresponding tRNAs. Thus,during the translation of AGGAGG tandems from foreign mRNA, +1 ribosomalframeshifts arise with a high frequency (about 50%) [Spanjaard et al.,Nuc. Acid Res., 18: 5031-5036 (1990)].

By immunoblotting, a similar pattern was obtained: all the bands seen onstained SDS-PAGE gel reacted with the MN-specific MAb M75 (FIG. 2B),indicating that all the protein bands are MN-specific. Also, that resultindicates that the binding site for MAb M75 is on the N-terminal part ofthe MN protein, which is not affected by frameshifts.

As shown in Example 8 below, the fusion protein pGEX-3X-MN was used inradioimmunoassays for MN-specific antibodies and for MN antigen.

MN Proteins and/or Polypeptides

The phrase "MN proteins and/or polypeptides" (MN proteins/polypeptides)is herein defined to mean proteins and/or polypeptides encoded by an MNgene or fragments thereof. Exemplary and preferred MN proteins accordingto this invention have the deduced amino acid sequences shown in FIGS.1A-1B and 15A-15C. Preferred MN proteins/polypeptides are those proteinsand/or polypeptides that have substantial homology with the MN proteinsshown in FIGS. 1A-1B and 15A-15C.

A "polypeptide" is a chain of amino acids covalently bound by peptidelinkages and is herein considered to be composed of 50 or less aminoacids. A "protein" is herein defined to be a polypeptide composed ofmore than 50 amino acids.

MN proteins exhibit several interesting features: cell membrane, and atthe same time, nuclear localization (similar to E6 protein of HPV16),cell density dependent expression in HeLa cells, correlation with thetumorigenic phenotype of HeLa×fibroblast somatic cell hybrids, andexpression in several human carcinomas among other tissues. Asdemonstrated herein, for example, in Example 14, MN protein can be founddirectly in tumor tissue sections but not in general in counterpartnormal tissues (exceptions noted infra in Example 14 as in normalstomach tissues). MN is also expressed sometimes in morphologicallynormal appearing areas of tissue specimens exhibiting dysplasia and/ormalignancy. Taken together, these features suggest a possibleinvolvement of MN in the regulation of cell proliferation,differentiation and/or transformation.

It can be appreciated that a protein or polypeptide produced by aneoplastic cell in vivo could be altered in sequence from that producedby a tumor cell in cell culture or by a transformed cell. Thus, MNproteins and/or polypeptides which have varying amino acid sequencesincluding without limitation, amino acid substitutions, extensions,deletions, truncations and combinations thereof, fall within the scopeof this invention. It can also be appreciated that a protein extantwithin body fluids is subject to degradative processes, such as,proteolytic processes; thus, MN proteins that are significantlytruncated and MN polypeptides may be found in body fluids, such as,sera. The phrase "MN antigen" is used herein to encompass MN proteinsand/or polypeptides.

It will further be appreciated that the amino acid sequence of MNproteins and polypeptides can be modified by genetic techniques. One ormore amino acids can be deleted or substituted. Such amino acid changesmay not cause any measurable change in the biological activity of theprotein or polypeptide and result in proteins or polypeptides which arewithin the scope of this invention.

The MN proteins and polypeptides of this invention can be prepared in avariety of ways according to this invention, for example, recombinantly,synthetically or otherwise biologically, that is, by cleaving longerproteins and polypeptides enzymatically and/or chemically. A preferredmethod to prepare MN proteins is by a recombinant means. A particularlypreferred method of recombinantly producing a MN protein is describedabove for the fusion protein pGEX-3X-MN.

Recombinant Production of MN Proteins and Polypeptides

A representative method to prepare the MN proteins shown in FIGS. 1A-1Band 15A-15C or fragments thereof would be to insert the appropriatefragment of the MN cDNA into an appropriate expression vector asexemplified above. A wide variety of host-cloning vector combinationsmay be usefully employed in cloning the MN DNA isolated as describedherein. For example, useful cloning vehicles may include chromosomal,nonchromosomal and synthetic DNA sequences such as various knownbacterial plasmids such as pBR322, other E. coli plasmids and theirderivatives and wider host range plasmids such as RP4, phage DNA, suchas, the numerous derivatives of phage lambda, e.g., NB989 and vectorsderived from combinations of plasmids and phage DNAs such as plasmidswhich have been modified to employ phage DNA expression controlsequences. The plasmid pGEX-3X is a preferred cloning vehicle.

Useful hosts may be eukaryotic or prokaryotic and include bacterialhosts such as E. coli and other bacterial strains, yeasts and otherfungi, animal or plant hosts such as animal or plant cells in culture,insect cells and other hosts. Of course, not all hosts may be equallyefficient. The particular selection of host-cloning vehicle combinationmay be made by those of skill in the art after due consideration of theprinciples set forth herein without departing from the scope of thisinvention.

The particular site chosen for insertion of the selected DNA fragmentinto the cloning vehicle to form a recombinant DNA molecule isdetermined by a variety of factors. These include size and structure ofthe protein or polypeptide to be expressed, susceptibility of thedesired protein or polypeptide to endoenzymatic degradation by the hostcell components and contamination by its proteins, expressioncharacteristics such as the location of start and stop codons, and otherfactors recognized by those of skill in the art.

The recombinant nucleic acid molecule containing the MN gene, fragmentthereof, or cDNA therefrom, may be employed to transform a host so as topermit that host (transformant) to express the structural gene orfragment thereof and to produce the protein or polypeptide for which thehybrid DNA encodes. The recombinant nucleic acid molecule may also beemployed to transform a host so as to permit that host on replication toproduce additional recombinant nucleic acid molecules as a source of MNnucleic acid and fragments thereof. The selection of an appropriate hostfor either of those uses is controlled by a number of factors recognizedin the art. These include, for example, compatibility with the chosenvector, toxicity of the co-products, ease of recovery of the desiredprotein or polypeptide, expression characteristics, biosafety and costs.

Where the host cell is a procaryote such as E. coli, competent cellswhich are capable of DNA uptake are prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ method bywell known procedures. Transformation can also be performed afterforming a protoplast of the host cell.

Where the host used is an eucaryote, transfection methods such as theuse of a calcium phosphate-precipitate, electroporation, conventionalmechanical procedures such as microinjection, insertion of a plasmidencapsulated in red blood cell ghosts or in liposomes, treatment ofcells with agents such as lysophosphatidyl-choline or use of virusvectors, or the like may be used.

The level of production of a protein or polypeptide is governed by threemajor factors: (1) the number of copies of the gene or DNA sequenceencoding for it within the cell; (2) the efficiency with which thosegene and sequence copies are transcribed and translated; and (3) thestability of the mRNA. Efficiencies of transcription and translation(which together comprise expression) are in turn dependent uponnucleotide sequences, normally situated ahead of the desired codingsequence. Those nucleotide sequences or expression control sequencesdefine, inter alia, the location at which an RNA polymerase interacts toinitiate transcription (the promoter sequence) and at which ribosomesbind and interact with the mRNA (the product of transcription) toinitiate translation. Not all such expression control sequences functionwith equal efficiency. It is thus of advantage to separate the specificcoding sequences for the desired protein from their adjacent nucleotidesequences and fuse them instead to known expression control sequences soas to favor higher levels of expression. This having been achieved, thenewly engineered DNA fragment may be inserted into a multicopy plasmidor a bacteriophage derivative in order to increase the number of gene orsequence copies within the cell and thereby further improve the yield ofexpressed protein.

Several expression control sequences may be employed. These include theoperator, promoter and ribosome binding and interaction sequences(including sequences such as the Shine-Dalgarno sequences) of thelactose operon of E. coli ("the lac system"), the correspondingsequences of the tryptophan synthetase system of E. coli ("the trpsystem"), a fusion of the trp and lac promoter ("the tac system"), themajor operator and promoter regions of phage lambda (O_(L) P_(L) andO_(R) P_(R),), and the control region of the phage fd coat protein. DNAfragments containing these sequences are excised by cleavage withrestriction enzymes from the DNA isolated from transducing phages thatcarry the lac or trp operons, or from the DNA of phage lambda or fd.Those fragments are then manipulated in order to obtain a limitedpopulation of molecules such that the essential controlling sequencescan be joined very close to, or in juxtaposition with, the initiationcodon of the coding sequence.

The fusion product is then inserted into a cloning vehicle fortransformation or transfection of the appropriate hosts and the level ofantigen production is measured. Cells giving the most efficientexpression may be thus selected. Alternatively, cloning vechiclescarrying the lac, trp or lambda P_(L) control system attached to aninitiation codon may be employed and fused to a fragment containing asequence coding for a MN protein or polypeptide such that the gene orsequence is correctly translated from the initiation codon of thecloning vehicle.

The phrase "recombinant nucleic acid molecule" is herein defined to meana hybrid nucleotide sequence comprising at least two nucleotidesequences, the first sequence not normally being found together innature with the second.

The phrase "expression control sequence" is herein defined to mean asequence of nucleotides that controls and regulates expression ofstructural genes when operatively linked to those genes.

Synthetic and Biologic Production of MN Proteins and Polypeptides

MN proteins and polypeptides of this invention may be prepared not onlyby recombinant means but also by synthetic and by other biologic means.Synthetic formation of the polypeptide or protein requires chemicallysynthesizing the desired chain of amino acids by methods well known inthe art. Exemplary of other biologic means to prepare the desiredpolypeptide or protein is to subject to selective proteolysis a longerMN polypeptide or protein containing the desired amino acid sequence;for example, the longer polypeptide or protein can be split withchemical reagents or with enzymes.

Chemical synthesis of a peptide is conventional in the art and can beaccomplished, for example, by the Merrifield solid phase synthesistechnique [Merrifield, J., Am. Chem. Soc., 85: 2149-2154 (1963); Kent etal., Synthetic Peptides in Biology and Medicine, 29 f.f. eds. Alitalo etal., (Elsevier Science Publishers 1985); and Haug, J. D., "PeptideSynthesis and Protecting Group Strategy", American BiotechnologyLaboratory, 5(1): 40-47 (January/February. 1987)].

Techniques of chemical peptide synthesis include using automatic peptidesynthesizers employing commercially available protected amino acids, forexample, Biosearch [San Rafael, Calif. (USA)] Models 9500 and 9600;Applied Biosystems, Inc. [Foster City, Calif. (USA)] Model 430; Milligen[a division of Millipore Corp.; Bedford, Mass. (USA)] Model 9050; and DuPont's RAMP (Rapid Automated Multiple Peptide Synthesis) [Du PontCompass, Wilmington, Del. (USA)].

Nucleic Acid Probes and Test Kits

Nucleic acid probes of this invention are those comprising sequencesthat are substantially complementary to the MN cDNA sequences shown inFIGS. 1A-1B and 15A-15C or to MN gene sequences. The phrase"substantially complementary" is defined herein to have the meaning asit is well understood in the art and, thus, used in the context ofstandard hybridization conditions. The stringency of hybridizationconditions can be adjusted to control the precision of complementarity.Exemplary are the stringent hybridization conditions used in Examples 12and 13.

Stringent hybridization conditions are considered herein to conform tostandard hybridization conditions understood in the art to be stringent.For example, it is generally understood that stringent conditionsencompass relatively low salt and/or high temperature conditions, suchas provided by 0.02 M to 0.15 M NaCl at temperatures of 50° C. to 70° C.Less stringent conditions, such as, 0.15 M to 0.9 M salt at temperaturesranging from 20° C. to 55° C. can be made more stringent by addingincreasing amounts of formamide, which serves to destabilize hybridduplexes as does increased temperature.

Exemplary stringent hybridization conditions are described in Examples12 and 13, infra; the hybridizations therein were carried out "in thepresence of 50% formamide at 42° C." [See Sambrook et al., MolecularCloning: A Laboratory Manual, pages 1.91 and 9.47-9.51 (Second Edition,Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989);Maniatis et al., Molecular Cloning: A Laboratory Manual, pages 387-389(Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y.; 1982);Tsuchiya et al., Oral Surgery, Oral Medicine, Oral Pathology, 71(6):721-725 (June 1991).]

Said nucleic acid probes are fragments of the isolated nucleic acidsequences that encode MN proteins or polypeptides according to thisinvention. Preferably those probes are composed of at least fiftynucleotides.

Said probes can be used to detect MN DNA and/or RNA, and thus can beused to test for the presence or absence of MN genes, andamplification(s), mutation(s) or genetic rearrangements of MN genes inthe cells of a patient. For example, overexpression of an MN gene may bedetected by Northern blotting using probes of this invention. Genealterations, as amplifications, translocations, inversions, anddeletions among others, can be detected by using probes of thisinvention for in situ hybridization to chromosomes from a patient'scells, whether in metaphase spreads or interphase nuclei. Southernblotting could also be used with the probes of this invention to detectamplifications or deletions of MN genes. Restriction Fragment LengthPolymorphism (RFLP) analysis using said probes is a preferred method ofdetecting gene alterations, mutations and deletions. Said probes canalso be used to identify MN proteins and/or polypeptides as well ashomologs or near homologs thereto by their hybridization to variousmRNAs transcribed from MN genes in different tissues.

Said probes thus can be useful diagnostically/prognostically. Saidprobes can be embodied in test kits, preferably with appropriate meansto enable said probes when hybridized to an appropriate MN gene or MNmRNA target to be visualized. Such samples include tissue specimens orsmears, body fluids and tissue and cell extracts.

Assays

Assays according to this invention are provided to detect and/orquantitate MN antigen or MN-specific antibodies in vertebrate samples,preferably mammalian samples, more preferably human samples. Suchsamples include tissue specimens, body fluids, tissue extracts and cellextracts. MN antigen may be detected by immunoassay, immunohistochemicalstaining, immunoelectron and scanning microscopy using immunogold amongother techniques.

Preferred tissue specimens to assay by immunohistochemical staininginclude cell smears, histological sections from biopsied tissues ororgans, and imprint preparations among other tissue samples. Such tissuespecimens can be variously maintained, for example, they can be fresh,frozen, or formalin-, alcohol- or acetone- or otherwise fixed and/orparaffin-embedded and deparaffinized. Biopsied tissue samples can be,for example, those samples removed by aspiration, bite, brush, cone,chorionic villus, endoscopic, excisional, incisional, needle,percutaneous punch, and surface biopsies, among other biopsy techniques.

Preferred cervical tissue specimens include cervical smears, conizationspecimens, histologic sections from hysterectomy specimens or otherbiopsied cervical tissue samples. Preferred means of obtaining cervicalsmears include routine swab, scraping or cytobrush techniques, amongother means. Papanicolaou-stained cervical smears (Pap smears) can bescreened by the methods of this invention, for example, forretrospective studies. Preferably, Pap smears would be decolorized andre-stained with labeled antibodies against MN antigen. Also archivalspecimens, for example, matched smears and biopsy and/or tumorspecimens, can be used for retrospective studies. Prospective studiescan also be done with matched specimens from patients that have a higherthan normal risk of exhibiting abnormal cervical cytopathology.

Preferred samples in which to assay MN antigen by, for example, Westernblotting or radioimmunoassay, are tissue and/or cell extracts. (Examples7 and 8 below are representative.) However, MN antigen may be detectedin body fluids, which can include among other fluids: blood, serum,plasma, semen, breast exudate, saliva, tears, sputum, mucous, urine,lymph, cytosols, ascites, pleural effusions, amniotic fluid, bladderwashes, bronchioalveolar lavages and cerebrospinal fluid. It ispreferred that the MN antigen be concentrated from a larger volume ofbody fluid before testing. Preferred body fluids to assay would dependon the type of cancer for which one was testing, but in generalpreferred body fluids would be breast exudate, pleural effusions andascites.

MN-specific antibodies can be bound by serologically active MNproteins/polypeptides in samples of such body fluids as blood, plasma,serum, lymph, mucous, tears, urine, spinal fluid and saliva; however,such antibodies are found most usually in blood, plasma and serum,preferably in serum. A representative assay to detect MN-specificantibodies is shown in Example 8 below wherein the fusion proteinpGEX-3X-MN is used. Correlation of the results from the assays to detectand/or quantitate MN antigen and MN-specific antibodies reactivetherewith, provides a preferred profile of the disease condition of apatient.

The assays of this invention are both diagnostic and/or prognostic,i.e., diagnostic/prognostic. The term "diagnostic/prognostic" is hereindefined to encompass the following processes either individually orcumulatively depending upon the clinical context: determining thepresence of disease, determining the nature of a disease, distinguishingone disease from another, forecasting as to the probable outcome of adisease state, determining the prospect as to recovery from a disease asindicated by the nature and symptoms of a case, monitoring the diseasestatus of a patient, monitoring a patient for recurrence of disease,and/or determining the preferred therapeutic regimen for a patient. Thediagnostic/prognostic methods of this invention are useful, for example,for screening populations for the presence of neoplastic orpre-neoplastic disease, determining the risk of developing neoplasticdisease, diagnosing the presence of neoplastic and/or pre-neoplasticdisease, monitoring the disease status of patients with neoplasticdisease, and/or determining the prognosis for the course of neoplasticdisease.

The present invention is useful for screening for the presence of a widevariety of neoplastic diseases including carcinomas, such as, mammary,urinary tract, ovarian, uterine, cervical, endometrial, squamous celland adenosquamous carcinomas; head and neck cancers; mesodermal tumors,such as, neuroblastomas and retinoblastomas; sarcomas, such asosteosarcomas and Ewing's sarcoma; and melanomas. Of particular interestare gynecological cancers including ovarian, uterine, cervical, vaginal,vulval and endometrial cancers, particularly ovarian, uterine cervicaland endometrial cancers. Also of particular interest are cancers of thebreast, of the stomach including esophagus, of the colon, of the kidney,of the prostate, of the liver, of the urinary tract including bladder,of the lung, and of the head and neck.

The invention provides methods and compositions for evaluating theprobability of the presence of malignant or pre-malignant cells, forexample, in a group of cells freshly removed from a host. Such an assaycan be used to detect tumors, quantitate their growth, and help in thediagnosis and prognosis of disease. The assays can also be used todetect the presence of cancer metastasis, as well as confirm the absenceor removal of all tumor tissue following surgery, cancer chemotherapyand/or radiation therapy. It can further be used to monitor cancerchemotherapy and tumor reappearance.

The presence of MN antigen or antibodies can be detected and/orquantitated using a number of well-defined diagnostic assays. Those inthe art can adapt any of the conventional immunoassay formats to detectand/or quantitate MN antigen and/or antibodies. Example 8 details theformat of a preferred diagnostic method of this invention--aradioimmunoassay. Immunohistochemical staining is another preferredassay format as exemplified in Example 14.

Many other formats for detection of MN antigen and MN-specificantibodies are, of course available. Those can be Western blots, ELISAs(enzyme-linked immunosorbent assays), RIAs (radioimmunoassay),competitive EIA or dual antibody sandwich assays, among other assays allcommonly used in the diagnostic industry. In such immunoassays, theinterpretation of the results is based on the assumption that theantibody or antibody combination will not cross-react with otherproteins and protein fragments present in the sample that are unrelatedto MN.

Representative of one type of ELISA test for MN antigen is a formatwherein a microtiter plate is coated with antibodies made to MNproteins/polypeptides or antibodies made to whole cells expressing MNproteins, and to this is added a patient sample, for example, a tissueor cell extract. After a period of incubation permitting any antigen tobind to the antibodies, the plate is washed and another set of anti-MNantibodies which are linked to an enzyme is added, incubated to allowreaction to take place, and the plate is then rewashed. Thereafter,enzyme substrate is added to the microtiter plate and incubated for aperiod of time to allow the enzyme to work on the substrate, and theadsorbance of the final preparation is measured. A large change inabsorbance indicates a positive result.

It is also apparent to one skilled in the art of immunoassays that MNproteins and/or polypeptides can be used to detect and/or quantitate thepresence of MN antigen in the body fluids, tissues and/or cells ofpatients. In one such embodiment, a competition immunoassay is used,wherein the MN protein/polypeptide is labeled and a body fluid is addedto compete the binding of the labeled MN protein/polypeptide toantibodies specific to MN protein/polypeptide. Such an assay can be usedto detect and/or quantitate MN antigen as described in Example 8.

In another embodiment, an immunometric assay may be used wherein alabeled antibody made to a MN protein or polypeptide is used. In such anassay, the amount of labeled antibody which complexes with theantigen-bound antibody is directly proportional to the amount of MNantigen in the sample.

A representative assay to detect MN-specific antibodies is a competitionassay in which labeled MN protein/polypeptide is precipitated byantibodies in a sample, for example, in combination with monoclonalantibodies recognizing MN proteins/polypeptides. One skilled in the artcould adapt any of the conventional immunoassay formats to detect and/orquantitate MN-specific antibodies. Detection of the binding of saidantibodies to said MN protein/polypeptide could be by many ways known tothose in the art, e.g., in humans with the use of anti-human labeledIgG.

An exemplary immunoassay method of this invention to detect and/orquantitate MN antigen in a vertebrate sample comprises the steps of:

a) incubating said vertebrate sample with one or more sets of antibodies(an antibody or antibodies) that bind to MN antigen wherein one set islabeled or otherwise detectable;

b) examining the incubated sample for the presence of immune complexescomprising MN antigen and said antibodies.

Another exemplary immunoassay method according to this invention is thatwherein a competition immunoassay is used to detect and/or quantitate MNantigen in a vertebrate sample and wherein said method comprises thesteps of:

a) incubating a vertebrate sample with one or more sets of MN-specificantibodies and a certain amount of a labeled or otherwise detectable MNprotein/polypeptide wherein said MN protein/polypeptide competes forbinding to said antibodies with MN antigen present in the sample;

b) examining the incubated sample to determine the amount oflabeled/detectable MN protein/polypeptide bound to said antibodies; and

c) determining from the results of the examination in step b) whether MNantigen is present in said sample and/or the amount of MN antigenpresent in said sample.

Once antibodies (including biologically active antibody fragments)having suitable specificity have been prepared, a wide variety ofimmunological assay methods are available for determining the formationof specific antibody-antigen complexes. Numerous competitive andnon-competitive protein binding assays have been described in thescientific and patent literature, and a large number of such assays arecommercially available. Exemplary immunoassays which are suitable fordetecting a serum antigen include those described in U.S. Pat Nos.3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987;3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; and 4,098,876.

Antibodies employed in assays may be labeled or unlabeled. Unlabeledantibodies may be employed in agglutination; labeled antibodies may beemployed in a wide variety of assays, employing a wide variety oflabels.

Suitable detection means include the use of labels such asradionuclides, enzymes, coenzymes, fluorescers, chemiluminescers,chromogens, enzyme substrates or co-factors, enzyme inhibitors, freeradicals, particles, dyes and the like. Such labeled reagents may beused in a variety of well known assays, such as radioimmunoassays,enzyme immunoassays, e.g., ELISA, fluorescent immunoassays, and thelike. See for example, U.S. Pat. Nos. 3,766,162; 3,791,932; 3,817,837;and 4,233,402.

Methods to prepare antibodies useful in the assays of the invention aredescribed below. The examples below detail representative assaysaccording to this invention.

Immunoassay Test Kits

The above outlined assays can be embodied in test kits to detect and/orquantitate MN antigen and/or MN-specific antibodies (includingbiologically active antibody fragments). Kits to detect and/orquantitate MN antigen can comprise MN protein(s)/polypeptides(s) and/orMN-specific antibodies, polyclonal and/or monoclonal. Suchdiagnostic/prognostic test kits can comprise one or more sets ofantibodies, polyclonal and/or monoclonal, for a sandwich format whereinantibodies recognize epitopes on the MN antigen, and one set isappropriately labeled or is otherwise detectable.

Test kits for an assay format wherein there is competition between alabeled (or otherwise detectable) MN protein/polypeptide and MN antigenin the sample, for binding to an antibody, can comprise the combinationof the labeled protein/polypeptide and the antibody in amounts whichprovide for optimum sensitivity and accuracy.

Test kits for MN-specific antibodies preferably compriselabeled/detectable MN proteins(s) and/or polypeptides(s), and maycomprise other components as necessary, for example, to perform apreferred assay as outlined in Example 8 below. Such test kits can haveother appropriate formats for conventional assays.

Preparation of MN-Specific Antibodies

The term "antibodies" is defined herein to include not only wholeantibodies but also biologically active fragments of antibodies,preferably fragments containing the antigen binding regions. Suchantibodies may be prepared by conventional methodology and/or by geneticengineering. Antibody fragments may be genetically engineered,preferably from the variable regions of the light and/or heavy chains(V_(H) and V_(L)), including the hypervariable regions, and still morepreferably from both the V_(H) and V_(L) regions. For example, the term"antibodies" as used herein comprehends polyclonal and monoclonalantibodies and biologically active fragments thereof including amongother possibilities "univalent" antibodies [Glennie et al., Nature, 295:712 (1982)]; Fab proteins including Fab' and F(ab')₂ fragments whethercovalently or non-covalently aggregated; light or heavy chains alone,preferably variable heavy and light chain regions (V_(H) and V_(L)regions), and more preferably including the hypervariable regions[otherwise known as the complementarity determining regions (CDRs) ofsaid V_(H) and V_(L) regions]; F_(c) proteins; "hybrid" antibodiescapable of binding more than one antigen; constant-variable regionchimeras; "composite" immunoglobulins with heavy and light chains ofdifferent origins; "altered" antibodies with improved specificity andother characteristics as prepared by standard recombinant techniques andalso by oligonucleotide-directed mutagenesis techniques[Dalbadie-McFarland et al., PNAS (USA), 79: 6409 (1982)].

It may be preferred for therapeutic and/or imaging uses that theantibodies be biologically active antibody fragments, preferablygenetically engineered fragments, more preferably genetically engineeredfragments from the V_(H) and/or V_(L) regions, and still more preferablycomprising the hypervariable regions thereof.

There are conventional techniques for making polyclonal and monoclonalantibodies well-known in the immunoassay art. Immunogens to prepareMN-specific antibodies include MN proteins and/or polypeptides,preferably purified, and MX-infected tumor line cells, for example,MX-infected HeLa cells, among other immunogens.

Anti-peptide antibodies are also made by conventional methods in the artas described in European Patent Publication No. 44,710 (published Jan.27, 1982). Briefly, such anti-peptide antibodies are prepared byselecting a peptide from an MN amino acid sequence as from FIGS. 1A-B or15A-15C, chemically synthesizing it, conjugating it to an appropriateimmunogenic protein and injecting it into an appropriate animal, usuallya rabbit or a mouse; then, either polyclonal or monoclonal antibodiesare made, the latter by the Kohler-Milstein procedure.

Besides conventional hybridoma technology, newer technologies can beused to produce antibodies according to this invention. For example, theuse of the polymerase chain reaction (PCR) to clone and express antibodyV-genes and phage display technology to select antibody genes encodingfragments with binding activities has resulted in the isolation ofantibody fragments from repertoires of PCR amplified V-genes usingimmunized mice or humans. [Marks et al., BioTechnology, 10: 779 (July1992) for references; Chiang et al., BioTechniques, 7(4): 360 (1989);Ward et al., Nature, 341: 544 (Oct. 12, 1989); Marks et al., J. Mol.Biol., 222: 581 (1991); Clackson et al., Nature, 352: (Aug. 15, 1991);and Mullinax et al., PNAS (USA), 87: 8095 (October 1990).

Descriptions of preparing antibodies, which term is herein defined toinclude biologically active antibody fragments, by recombinanttechniques can be found in U.S. Pat. No. 4,816,567 (issued Mar. 28,1989); European Patent Application Publication Number (EP) 338,745(published Oct. 25, 1989); EP 368,684 (published Jun. 16, 1990); EP239,400 (published Sep. 30, 1987); WO 90/14424 (published Nov. 29,1990); WO 90/14430 (published May 16, 1990); Huse et al., Science, 246:1275 (Dec. 8, 1989); Marks et al., BioTechnology, 10: 779 (July 1992);La Sastry et al., PNAS (USA), 86: 5728 (Aug. 1989); Chiang et al.,BioTechniques, 7(40): 360 (1989); Orlandi et al., PNAS (USA), 86: 3833(May 1989); Ward et al. Nature, 341: 544 (Oct. 12, 1989); Marks et al.,J. Mol. Biol., 222: 581 (1991); and Hoogenboom et al., Nucleic AcidsRes., 19(15): 4133 (1991).

Preparation of Monoclonal Antibodies

Monoclonal antibodies for use in the assays of this invention may beobtained by methods well known in the art for example, Galfre andMilstein, "Preparation of Monoclonal Antibodies: Strategies andProcedures," in Methods in Enzymology: Immunochemical Techniques, 73:1-46 [Langone and Vanatis (eds); Academic Press (1981)]; and in theclassic reference, Milstein and Kohler, Nature, 256: 495-497 (1975).

Although the representative hybridoma of this invention is formed by thefusion of murine cell lines, human/human hybridomas [Olsson et al., PNAS(USA), 77: 5429 (1980)] and human/murine hybridomas [Schlom et al., PNAS(USA), 77: 6841 (1980); Shearman et al. J. Immunol., 146: 928-935(1991); and Gorman et al., PNAS (USA), 88: 4181-4185 (1991)] can also beprepared among other possibilities. Such humanized monoclonal antibodieswould be preferred monoclonal antibodies for therapeutic and imaginguses.

Monoclonal antibodies specific for this invention can be prepared byimmunizing appropriate mammals, preferably rodents, more preferablyrabbits or mice, with an appropriate immunogen, for example,MaTu-infected HeLa cells or MN proteins/polypeptides attached to acarrier protein if necessary. The production of hybridoma VU-M75 whichsecretes MAb M75 is exemplary and described below. MAb M75 serves toidentify MN proteins/polypeptides in various laboratory diagnostictests, for example, in tumor cell cultures or in clinical samples. Alsoproduced by the method described for producing MAb M75 (isotype IgG2B)were MAbs M16 (isotype IgG2A) and M67 (isotype IgG1).

MAb M75

Monoclonal antibody M75 (MAb M75) is produced by mouse lymphocytichybridoma VU-M75, which was initially deposited in the Collection ofHybridomas at the Institute of Virology, Slovak Academy of Sciences(Bratislava, Czechoslovakia) and was deposited under ATCC Designation HB11128 on Sep. 17, 1992 at the American Type Culture Collection (ATCC) inManassas, Va. (USA).

Hybridoma VU-M75 was produced according to the procedure described inGerhard, W., "Fusion of cells in suspension and outgrowth of hybrids inconditioned medium," In: Monoclonal Antibodies. Hybridomas: A NewDimension in Biological Analysis, page 370 [Kennet et al. (eds.); PlenumN.Y. (USA)]. BALB/C mice were immunized with MaTu-infected HeLa cells,and their spleen cells were fused with myeloma cell line NS-0. Tissueculture media from the hybridomas were screened for monoclonalantibodies, using as antigen the p58 immunoprecipitated from cellextracts of MaTu-infected HeLa with rabbit anti-MaTu serum and proteinA-Staphylococcus aureus cells (SAC) [Zavada and Zavadova, Arch. Virol.,118 189-197 (1991)], and eluted from SDS-PAGE gels. Monoclonalantibodies were purified from TC media by affinity chromatography onprotein A-Sepharose [Harlow and Lane, "Antibodies: A Laboratory Manual,"Cold Spring Harbor, Cold Spring Harbor, N.Y. (USA); 1988].

The monoclonal antibodies useful according to this invention to identifyMN proteins/polypeptides can be labeled in any conventional manner, forexample, with enzymes such as horseradish peroxidase (HRP), fluorescentcompounds, or with radioactive isotopes such as, ¹²⁵ I, among otherlabels. A preferred label, according to this invention is ¹²⁵ I, and apreferred method of labeling the antibodies is by using chloramine-T[Hunter, W. M., "Radioimmunoassay," In: Handbook of ExperimentalImmunology, pp. 14.1-14.40 (D. W. Weir ed.; Blackwell,Oxford/London/Edinburgh/Melbourne; 1978)].

MAb H460

Monoclonal antibody H460 (MAb H460) was prepared in a manner similar tothat for MAb M75 except that the mice were immunized with HeLa cellsuninfected with MaTu, and lymphocytes of the mice rather than spleencells were fused with cells from myeloma cell line NS-0. MAb H460 reactsabout equally with any human cells.

Therapeutic Use of MN-Specific Antibodies

The MN-specific antibodies of this invention, monoclonal and/orpolyclonal, preferably monoclonal, more preferably MAb M75, may be usedtherapeutically in the treatment of neoplastic and/or pre-neoplasticdisease, either alone or in combination with chemotherapeutic drugs ortoxic agents, such as ricin A. Further preferred for therapeutic usewould be biologically active antibody fragments as described herein.Also preferred MN-specific antibodies for such therapeutic uses would behumanized monoclonal antibodies.

The MN-specific antibodies can be administered in a therapeuticallyeffective amount, preferably dispersed in a physiologically acceptable,nontoxic liquid vehicle.

Imaging Use of Antibodies

Further, the MN-specific antibodies of this invention when linked to animaging agent, such as a radionuclide, can be used for imaging.Biologically active antibody fragments or humanized monoclonalantibodies, may be preferred for imaging use.

A patient's neoplastic tissue can be identified as, for example, sitesof transformed stem cells, of tumors and locations of any metastases.Antibodies, appropriately labeled or linked to an imaging agent, can beinjected in a physiologically acceptable carrier into a patient, and thebinding of the antibodies can be detected by a method appropriate to thelabel or imaging agent, for example, by scintigraphy.

Antisense MN Nucleic Acid Sequences

MN genes are herein considered putative oncogenes and the proteinsencoded thereby are considered to be putative oncoproteins. Antisensenucleic acid sequences substantially complementary to mRNA transcribedfrom MN genes, as represented by the antisense oligodeoxynucleotides(ODNs) of Example 11, infra, can be used to reduce or prevent expressionof the MN gene. [Zamecnick, P. C., "Introduction: Oligonucleotide BaseHybridization as a Modulator of Genetic Message Readout," pp. 1-6,Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS,(Wiley-Liss, Inc., New York, N.Y., USA; 1991); Wickstrom, E., "AntisenseDNA Treatment of HL-60 Promyelocytic Leukemia Cells: TerminalDifferentiation and Dependence on Target Sequence," pp. 7-24, id.;Leserman et al., "Targeting and Intracellular Delivery of AntisenseOligonucleotides Interfering with Oncogene Expression," pp. 25-34, id.;Yokoyama, K., "Transcriptional Regulation of c-myc Proto-oncogene byAntisense RNA," pp. 35-52, id.; van den Berg et al., "Antisense fosoligodeoxyribonucleotides Suppress the Generation of ChromosomalAberrations," pp. 63-70, id.; Mercola, D., "Antisense fos and fun RNA,"pp. 83-114, id.; Inouye, Gene, 72: 25-34 (1988); Miller and Ts'o, Ann.Reports Med. Chem., 23: 295-304 (1988); Stein and Cohen, Cancer Res.,48: 2659-2668 (1988); Stevenson and Inversen, J. Gen. Virol., 70:2673-2682 (1989); Goodchild, "Inhibition of Gene Expression byOligonucleotides," pp. 53-77, Oligodeoxynucleotides: AntisenseInhibitors of Gene Expression (Cohen, J. S., ed; CRC Press, Boca Raton,Fla., USA; 1989); Dervan et al., "Oligonucleotide Recognition ofDouble-helical DNA by Triple-helix Formation," pp. 197-210, id.;Neckers, L. M., "Antisense Oligodeoxynucleotides as a Tool for StudyingCell Regulation: Mechanisms of Uptake and Application to the Study ofOncogene Function," pp. 211-232, id.; Leitner et al., PNAS (USA), 87:3430-3434 (1990); Bevilacqua et al., PNAS (USA), 85: 831-835 (1988);Loke et al. Curr. Top. Microbiol. Immunol., 141: 282-288 (1988); Sarinet al., PNAS (USA), 85: 7448-7451 (1988); Agrawal et al., "AntisenseOligonucleotides: A Possible Approach for Chemotherapy and AIDS,"International Union of Biochemistry Conference on Nucleic AcidTherapeutics (Jan. 13-17, 1991; Clearwater Beach, Fla., USA); Armstrong,L., Ber. Week, pp. 88-89 (Mar. 5, 1990); and Weintraub et al., Trends,1: 22-25 (1985).] Such antisense nucleic acid sequences, preferablyoligonucleotides, by hybridizing to the MN mRNA, particularly in thevicinity of the ribosome binding site and translation initiation point,inhibits translation of the mRNA. Thus, the use of such antisensenucleic acid sequences may be considered to be a form of cancer therapy.

Preferred antisense oligonucleotides according to this invention aregene-specific ODNs or oligonucleotides complementary to the 5' end of MNmRNA. Particularly preferred are the 29-mer ODN1 and 19-mer ODN2 forwhich the sequences are provided in Example 11, infra. Those antisenseODNs are representative of the many antisense nucleic acid sequencesthat can function to inhibit MN gene expression. Ones of ordinary skillin the art could determine appropriate antisense nucleic acid sequences,preferably antisense oligonucleotides, from the nucleic acid sequencesof FIGS. 1A-1B and 15A-15C.

Vaccines

It will be readily appreciated that MN proteins and polypeptides of thisinvention can be incorporated into vaccines capable of inducingprotective immunity against neoplastic disease and a dampening effectupon tumorigenic activity. Efficacy of a representative MN fusionprotein pGEX-3X-MN as a vaccine in a rat model is shown in Example 15.

MN proteins and/or polypeptides may be synthesized or preparedrecombinantly or otherwise biologically, to comprise one or more aminoacid sequences corresponding to one or more epitopes of the MN proteinseither in monomeric or multimeric form. Those proteins and/orpolypeptides may then be incorporated into vaccines capable of inducingprotective immunity. Techniques for enhancing the antigenicity of suchpolypeptides include incorporation into a multimeric structure, bindingto a highly immunogenic protein carrier, for example, keyhole limpethemocyanin (KLH), or diptheria toxoid, and administration in combinationwith adjuvants or any other enhancers of immune response.

Preferred MN proteins/polypeptides to be used in a vaccine according tothis invention would be genetically engineered MN proteins. A preferredrecombinant MN protein is the fusion protein pGEX-3X-MN, producedaccording to this invention.

A preferred exemplary use of such a vaccine of this invention would beits administration to patients whose MN-carrying primary cancer had beensurgically removed. The vaccine may induce active immunity in thepatients and prevent recidivism or metastasis.

It will further be appreciated that anti-idiotype antibodies toantibodies to MN proteins/polypeptides are also useful as vaccines andcan be similarly formulated.

An amino acid sequence corresponding to an epitope of an MNprotein/polypeptide either in monomeric or multimeric form may also beobtained by chemical synthetic means or by purification from biologicalsources including genetically modified microorganisms or their culturemedia [See Lerner, "Synthetic Vaccines", Sci. Am. 248(2): 66-74 (1983)].The protein/polypeptide may be combined in an amino acid sequence withother proteins/polypeptides including fragments of other proteins, asfor example, when synthesized as a fusion protein, or linked to otherantigenic or non-antigenic polypeptides of synthetic or biologicalorigin. In some instances, it may be desirable to fuse a MN protein orpolypeptide to an immunogenic and/or antigenic protein or polypeptide,for example, to stimulate efficacy of a MN-based vaccine.

The term "corresponding to an epitope of an MN protein/polypeptide" willbe understood to include the practical possibility that, in someinstances, amino acid sequence variations of a naturally occurringprotein or polypeptide may be antigenic and confer protective immunityagainst neoplastic disease and/or anti-tumorigenic effects. Possiblesequence variations include, without limitation, amino acidsubstitutions, extensions, deletions, truncations, interpolations andcombinations thereof. Such variations fall within the contemplated scopeof the invention provided the protein or polypeptide containing them isimmunogenic and antibodies elicited by such a polypeptide or proteincross-react with naturally occurring MN proteins and polypeptides to asufficient extent to provide protective immunity and/or anti-tumorigenicactivity when administered as a vaccine.

Such vaccine compositions will be combined with a physiologicallyacceptable medium, including immunologically acceptable diluents andcarriers as well as commonly employed adjuvants such as Freund'sComplete Adjuvant, saponin, alum, and the like. Administration would bein immunologically effective amounts of the MN proteins or polypeptides,preferably in quantities providing unit doses of from 0.01 to 10.0micrograms of immunologically active MN protein and/or polypeptide perkilogram of the recipient's body weight. Total protective doses mayrange from 0.1 to about 100 micrograms of antigen.

Routes of administration, antigen dose, number and frequency ofinjections are all matters of optimization within the scope of theordinary skill in the art.

The following examples are for purposes of illustration only and notmeant to limit the invention in any way.

Materials and Methods

The following materials and methods were used in the Examples below.

MaTu-Infected and Uninfected HeLa Cells

MaTu agent [Zavada et al., Nature New Biol., 240: 124-125 (1972); Zavadaet al., J. Gen. Virol, 24: 327-337 (1974)] was from original "MaTu"cells [Widmaier et al., Arch. Geschwulstforsch, 44: 1-10 (1974)]transferred into our stock of HeLa by cocultivation with MaTu cellstreated with mitomycin C, to ensure that control and MaTu-infected cellswere comparable. MaTu cells were incubated for 3 hours at 37° C. inmedia with 5 μg/ml of mitomycin C [Calbiochem, LaJolla, Calif. (USA)].Mixed cultures were set to 2×10⁵ of mitomycin C-treated cells and 4×10⁵of fresh recipient cells in 5 ml of medium. After 3 days they were firstsubcultured and further passaged 1-2 times weekly.

Control HeLa cells were the same as those described in Zavada et al.,Nature New Biol. 240: 124-125 (1972).

Sera

Human sera from cancer patients, from patients suffering with variousnon-tumor complaints and from healthy women were obtained from theClinics of Obstetrics and Gynaecology at the Postgraduate MedicalSchool, Bratislava, Czechoslovakia.

Human sera KH was from a fifty year old mammary carcinoma patient,fourteen months after resection. That serum was one of two sera out of401 serum samples that contained neutralizing antibodies to theVSV(MaTU) pseudotype as described in Zavada et al., Nature New Biology,240: 124-125 (1972). Serum L8 was from a patient with Paget's disease.Serum M7 was from a healthy donor.

Rabbit anti-MaTu serum was prepared by immunizing a rabbit three timesat intervals of 30 days with 1-5×10⁷ viable MaTu infected HeLa cells.

RIP and PAGE

RIP and PAGE were performed essentially as described in Zavada andZavadova, Arch. Virol., 118: 189-197 (1991), except that in theexperiments described herein [³⁵ S]methionine (NEN), 10 μCi/ml ofmethionine-free MEM medium, supplemented with 2% FCS and 3% complete MEMwere used. Confluent petri dish cultures of cells were incubatedovernight in that media.

For RIP, the SAC procedure [Kessler, J. Immunol., 115: 1617-1624 (1975)]was used. All incubations and centrifugations were performed at 0-4° C.Cell monolayers were extracted with RIPA buffer (0.14 M NaCl, 7.5 mMphosphate buffer, pH 7.2, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM phenylmethylsulfonyl fluoride and Trasylol). To reduce non-specificreactions, antisera were preabsorbed with foetal calf serum [Barbacid etal., PNAS (USA), 77: 1617-1621 (1980)] and antigenic extracts with SAC.

For PAGE (under reducing conditions) we used 10% gels with SDS [Laemmli,Nature, 227: 680-685 (1970)]. As reference marker proteins served theSigma kit (product MW-SDS-200). For fluorography we used salicylate[Heegaard et al., Electrophoresis, 5: 263-269 (1984)].

Immunoblots

Immunoblotting used as described herein follows the method of Towbin etal., PNAS (USA), 76: 4350-4354 (1979). The proteins were transferredfrom the gels onto nitrocellulose [Schleicher and Schuell; DasselGermany; 0.45 μm porosity] in Laemmli electrode buffer diluted 1:10 withdistilled water, with no methanol or SDS. The transfer was for 2 1/2hours at 1.75 mA/cm². The blots were developed with ¹²⁵ I-labeled MAbsand autoradiography was performed using intensifying screens, with X-rayfilms exposed at -70° C.

In extracts from cell cultures containing only small amounts of MNantigen, we concentrated the antigen from 0.5 or 1 ml of an extract byadding 50 μl of a 10% SAC suspension, pre-loaded with MAb M75. Thismethod allowed the concentration of MN antigen even from clinicalspecimens, containing human IgG; preliminary control experiments showedthat such a method did not interfere with the binding of the MN antigento SAC-adsorbed M75. Tissue extracts were made by grinding the tissuewith a mortar and pestle and sand (analytical grade). To the homogenateswas added RIPA buffer, 10:1 (volume to weight) of original tissue. Theextracts were clarified for 3 minutes on an Eppendorf centrifuge.

EXAMPLE 1 Immunofluorescence of MaTu-Specific Antigens

Immunofluorescence experiments were performed on control andMaTu-infected HeLa cells with monoclonal antibodies, prepared asdescribed above, which are specific for MaTu-related antigens.FITC-conjugated anti-mouse IgG was used to detect the presence of themonoclonal antibodies. Staining of the cells with Giemsa revealed noclear differences between control and MaTu-infected HeLa cells.

MAbs, which in preliminary tests proved to be specific for MaTu-relatedantigens, showed two different reactivities in immunofluorescence. Arepresentative of the first group, MAb M67, gave a granular cytoplasmicfluorescence in MaTu-infected HeLa, which was only seen in cells fixedwith acetone; living cells showed no fluorescence. MAb M16 gave the sametype of fluorescence. With either M67 or M16, only extremely weak"background" fluorescence was seen in control HeLa cells.

Another MAb, M75, showed a granular membrane fluorescence on livingMaTu-infected cells and a granular nuclear fluorescence in acetone-fixedcells. However, M75 sometimes showed a similar, although much weaker,fluorescence on uninfected HeLa cells. A relationship was observed basedupon the conditions of growth: in HeLa cells uninfected with MaTu, bothtypes of fluorescence with MAb M75 were observed only if the cells weregrown for several passages in dense cultures, but not in sparse ones.

The amount of M75-reactive cell surface antigen was analyzedcytofluorometrically and was dependent on the density of the cellcultures and on infection with MaTu. Control and MaTu infected HeLacells were grown for 12 days in dense or sparse cultures. The cells werereleased with Versene (EDTA), and incubated with MAb M75 or with no MAb,and subsequently incubated with FITC-conjugated anti-mouse IgG. Theintensity of fluorescence was measured.

It appeared that the antigen binding MAb M75 is inducible: it was foundto be absent in control HeLa grown in sparse culture, and to be inducedeither by the growth of HeLa in dense culture or by infection with MaTu.Those two factors were found to have an additive or synergistic effect.Those observations indicated along with other results described hereinthat there were two different agents involved: exogenous, transmissibleMX, reactive with M67, and endogenous, inducible MN, detected by MAbM75.

EXAMPLE 2 Immunoblot Analysis of Protein(s) Reactive with MAb M75

To determine whether MAb M75 reacts with the same protein in bothuninfected and MaTu-infected HeLa, and to determine the molecular weightof the protein, extracts of those cells were analyzed by PAGE andimmunoblotting (as described above). HeLa cells uninfected orMaTu-infected, that had been grown for 12 days in dense or sparsecultures, were seeded in 5-cm petri dishes, all variants at 5×10⁵ cellsper dish. Two days later, the cells were extracted with RIPA buffer(above described), 200 μl/dish. The extracts were mixed with 2×concentrated Laemmli sample buffer containing 6% mercaptoethanol andboiled for five minutes. Proteins were separated by SDS-PAGE and blottedon nitrocellulose. The blots were developed with ¹²⁵ I-labeled MAb M75and autoradiography.

MAb M75 reacted with two MN-specific protein bands of 54 kd and 58 kd,which were the same in uninfected HeLa grown at high density and inMaTu-infected HeLa, evidencing that M75 recognizes the same protein(s)in both uninfected and MaTu-infected HeLa cells. Consistent with thecytofluorometric results, the amount of the antigen depended both oncell density and on infection with MaTu, the latter being a much morepotent inducer of p54/58N.

EXAMPLE 3 Radioimmunoassay of MaTu-Specific Antigens In Situ

In contrast to the results with M75, the other MAb, M67, appeared to bespecific for the exogenous, transmissible agent MX. With M67 we observedno immunofluorescence in control HeLa, regardless of whether the cellswere grown in dense or in sparse culture. That difference was clearlyevidenced in radioimmunoassay experiments wherein ¹²⁵ I-labeled MAbs M67and M75 were used.

For such experiments, parallel cultures of uninfected and MaTu-infectedcells were grown in dense or sparse cultures. The cultures were eitherlive (without fixation), or fixed (with methanol for five minutes andair-dried). The cultures were incubated for two hours in petri disheswith the ¹²⁵ I-labeled MAbs, 6×10⁴ cpm/dish. Afterward, the cultureswere rinsed four times with PBS and solubilized with 1 ml/dish of 2 NNaOH, and the radioactivity was determined on a gamma counter.

The simple radioimmunoassay procedure of this example was performeddirectly in petri dish cultures. Sixteen variants of theradioimmunoassay enabled us to determine whether the MX and MN antigensare located on the surface or in the interior of the cells and how theexpression of those two antigens depends on infection with MaTu and onthe density, in which the cells had been grown before the petri disheswere seeded. In live, unfixed cells only cell surface antigens can bindthe MAbs. In those cells, M67 showed no reaction with any variant of thecultures, whereas M75 reacted in accord with the results of Examples 1and 2 above.

Fixation of the cells with methanol made the cell membrane permeable tothe MAbs: M67 reacted with HeLa infected with MaTu, independently ofprevious cell density, and it did not bind to control HeLa. MAb M75 inmethanol-fixed cells confirmed the absence of corresponding antigen inuninfected HeLa from sparse cultures and its induction both by growth indense cultures and by infection with MaTu.

EXAMPLE 4 Identification of MaTu Components Reactive with Animal Sera orAssociated with VSV Virions

Immunoblot analyses of MaTu-specific proteins from RIPA extracts fromuninfected or MaTu-infected HeLa and from purified VSV reproduced incontrol or in MaTu-infected HeLa, identified which of the antigens, p58Xor p54/58N, were radioimmunoprecipitated with animal sera, and which ofthem was responsible for complementation of VSV mutants and for theformation of pseudotype virions. Details concerning the procedures canbe found in Pastorekova et al., Virology, 187: 620-626 (1992).

The serum of a rabbit immunized with MaTu-infected HeLaimmunoprecipitated both MAb M67- and MAb M75-reactive proteins (bothp58X and p54/58N), whereas the "spontaneously" immune sera of normalrabbit, sheep or leukemic cow immunoprecipitated only the M67-reactiveprotein (p58X). On the other hand, in VSV reproduced in MaTu-infectedHeLa cells and subsequently purified, only the M75-reactive bands ofp54/58N were present. Thus, it was concluded that MX and MN areindependent components of MaTu, and that it was p54/58N thatcomplemented VSV mutants and was assembled into pseudotype virions.

As shown in FIGS. 6A-6B discussed below in Example 5, MX antigen wasfound to be present in MaTu-infected fibroblasts. In Zavada and Zavadova(1991), it was reported that a p58 band from MX-infected fibroblastscould not be detected by RIP with rabbit anti-MaTu serum. That serumcontains more antibodies to MX than to MN antigen. The discrepancy canbe explained by the extremely slow spread of MX in infected cultures.The results reported in Zavada and Zavadova (1991) were from fibroblaststested 6 weeks after infection, whereas the later testing was 4 monthsafter infection. We have found by immunoblots that MX can be firstdetected in both H/F-N and H/F-T hybrids after 4 weeks, in HeLa cellsafter six weeks and in fibroblasts only 10 weeks after infection.

EXAMPLE 5 Expression of MN- and MX- Specific Proteins

FIGS. 6A-6B graphically illustrates the expression of MN- and MX-specific proteins in human fibroblasts, in HeLa cells and in H/F-N andH/F-T hybrid cells, and contrasts the expression in MX-infected anduninfected cells. Cells were infected with MX by co-cultivation withmitomycin C-treated MX-infected HeLa. The infected and uninfected cellswere grown for three passages in dense cultures. About four months afterinfection, the infected cells concurrently with uninfected cells weregrown in petri dishes to produce dense monolayers.

A radimmunoassay was performed directly in confluent petri dish (5 cm)culture of cells, fixed with methanol essentially as described inExample 3, supra. The monolayers were fixed with methanol and treatedwith ¹²⁵ I-labeled MAbs M67 (specific for exogenous MX antigen) or M75(specific for endogenous MN antigen) at 6×10⁴ cpm/dish. The boundradioactivity was measured; the results are shown in FIGS. 6A-6B.

FIGS. 6A-6B shows that MX was transmitted to all four cell lines tested,that is, to human embryo fibroblasts, to HeLa and to both H/F-N andH/F-T hybrids; at the same time, all four uninfected counterpart celllines were MX-negative (top graph of FIG. 6A). MN antigens are shown tobe present in both MX-infected and uninfected HeLa and H/F-T cells, butnot in the fibroblasts (bottom graph of FIG. 6B). No MN antigen wasfound in the control H/F-N, and only a minimum increase over backgroundof MN antigen was found in MaTu infected H/F-N. Thus, it was found thatin the hybrids, expression of MN antigen very strongly correlates withtumorigenicity.

Those results were consistent with the results obtained byimmunoblotting as shown in FIG. 7. The MN-specific twin protein p54/58Nwas detected in HeLa cell lines (both our standard type, that is, HeLaK, and in the Stanbridge mutant HeLa, that is, D98/AH.2 shown as HeLa S)and in tumorigenic H/F-T; however, p54/58N was not detected in thefibroblasts nor in the non-tumorigenic H/F-N even upon deliberately longexposure of the film used to detect radioactivity. Infection of the HeLacells with MX resulted in a strong increase in the concentration of thep54/58N protein(s).

The hybrid cells H/F-N and H/F-T were constructed by Eric J. Stanbridge[Stanbridge et al., Somatic Cell Genetics, 7: 699-712 (1981); andStanbridge et al., Science, 215: 252-259 (1982)]. His original hybrid,produced by the fusion of a HeLa cell and a human fibroblast was nottumorigenic in nude mice, although it retained some properties oftransformed cells, for example, its growth on soft agar. Rare segregantsfrom the hybrid which have lost chromosome 11 are tumorigenic. The mostlikely explanation for the tumorigenicity of those segregants is thatchromosome 11 contains a suppressor gene (an antioncogene), which blocksthe expression of a as yet unknown oncogene. The oncoprotein encoded bythat oncogene is critical for the capacity of the H/F hybrids to producetumors in nude mice. Since the p54/58N protein shows a correlation withthe tumorigenicity of H/F hybrids, it is a candidate for that putativeoncoprotein.

EXAMPLE 6 Immunoblots of MN Antigen from Human Tumor Cell Cultures andfrom Clinical Specimens of Human Tissues

The association of MN antigen with tumorigenicity in the H/F hybridcells as illustrated by Example 5 prompted testing for the presence ofMN antigen in other human tumor cell cultures and in clinical specimens.Preliminary experiments indicated that the concentration of MN antigenin the extracts from other human tumor cell cultures was lower than inHeLa; thus, it was realized that long exposure of the autoradiographswould be required. Therefore, the sensitivity of the method wasincreased by the method indicated under Materials and Methods:Immunoblotting, supra, wherein the MN antigen was concentrated byprecipitation with MAb M75-loaded SAC.

FIG. 8 shows the immunoblots wherein lane A, a cell culture extract fromMX-infected HeLa cells was analysed directly (10 μl per lane) whereasthe antigens from the other extracts (lanes B-E) were each concentratedfrom a 500 μl extract by precipitation with MAb M75 and SAC.

FIG. 8 indicates that two other human carcinoma cell lines containMN-related proteins--T24 (bladder carcinoma; lane C) and T47D (mammarycarcinoma; lane D). Those cells contain proteins which react with MAbM75 that under reducing conditions have molecular weights of 54 kd and56 kd, and under non-reducing conditions have a molecular weight ofabout 153 kd. The intensity of those bands is at least ten times lowerthan that for the p54/58N twin protein from HeLa cells.

An extremely weak band at approximately 52 kd could be seen underreducing conditions from extracts from human melanoma cells (SK-Mel1477;lane E), but no bands for human fibroblast extracts (lane B) couldbe seen either on the reducing or non-reducing gels.

FIG. 9 shows immunoblots of human tissue extracts including surgicalspecimens as compared to a cell extract from MX-infected HeLa (lane A).The MN-related antigen from all the extracts but for lane A (analyseddirectly at 10 μl per lane) was first concentrated from a 1 ml extractas explained above. MN proteins were found in endometrial (lanes D andM), ovarian (lanes E and N) and in uterine cervical (lane 0) carcinomas.In those extracts MN-related proteins were found in three bands havingmolecular weights between about 48 kd and about 58 kd. AnotherMN-related protein was present in the tissue extract from a mammarypapilloma; that protein was seen as a single band at about 48 kd (laneJ).

Clearly negative were the extracts from full-term placenta (lane B),normal mammary gland (lane K), hyperplastic endometrium (lane L), normalovaries (lane H), and from uterine myoma (lane I). Only extremelyslightly MN-related bands were seen in extracts from trophoblasts (lanesF and G) and from melanoma (lane P).

The observations that antigen related to p54/58N was expressed inclinical specimens of several types of human carcinomas but not ingeneral in normal tissues of the corresponding organs (exceptionsdelineated in Example 14) further strengthened the association of MNantigen with tumorigenesis. However, it should be noted that for humantumors, a normal tissue is never really an adequate control in thattumors are believed not to arise from mature, differentiated cells, butrather from some stem cells, capable of division and of differentiation.In body organs, such cells may be quite rare.

EXAMPLE 7 MN Antigen in Animal Cell Lines

Since the MN gene is present in the chromosomal DNA of all vertebratespecies that were tested, MN-related antigen was searched for also incell lines derived from normal tissues and from tumors of several animalspecies. MN-related protein was found in two rat cell lines: one of themwas the XC cell line derived from rat rhabdomyosarcoma induced with Roussarcoma virus; the other was the Rat2-Tk⁻ cell line. In extracts fromboth of those rat cell lines, a single protein band was found on theblots: its molecular weight on blots produced from a reducing gel andfrom a non-reducing gel was respectively 53.5 kd and 153 kd. FIG. 10shows the results with Rat2-Tk⁻ cell extracts (lane B), compared withextracts from MX-infected HeLa (lane A); the concentration of MN antigenin those two cell lines is very similar. The extracts were analyseddirectly (40 μl per lane).

MN-related protein from XC cells showed the same pattern as for Rat2-Tk⁻cells both under reducing and non-reducing conditions, except that itsconcentration was about 30× lower. The finding of a MN-relatedprotein--p53.5N--in two rat cell lines (FIGS. 10 and 12) provides thebasis for a model system.

None of the other animal cell lines tested contained detectable amountsof MN antigen, even when the highly sensitive immunoblot technique inwhich the MN antigens are concentrated was used. The MN-negative cellswere: Vero cells (African green monkey); mouse L cells; mouse NIH-3T3cells normal, infected with Moloney leukemia virus, or transformed withHarvey sarcoma virus; GR cells (mouse mammary tumor cells induced withMTV), and NMG cells (normal mouse mammary gland).

EXAMPLE 8 Radioimmunoassays in Liquid Phase Using Recombinant MN Proteinfor MN-Specific Antibodies and for MN Antigen

The genetically engineered MN protein fused with glutathioneS-transferase--pGEX-3X-MN--prepared and purified as described above waslabeled with ¹²⁵ I by the chloramine T method [Hunter (1978)]. Thepurified protein enabled the development of a quantitative RIA forMN-specific antibodies as well as for MN antigens. All dilutions ofantibodies and of antigens were prepared in RIPA buffer (1% TRITON X-100and 0.1% sodium deoxycholate in PBS--phosphate buffered saline, pH 7.2),to which was added 1% of foetal calf serum (FCS). Tissue and cellextracts were prepared in RIPA buffer containing 1 mMphenylmethylsulfonylfluoride and 200 trypsin inhibiting units ofTrasylol (aprotinin) per ml, with no FCS. ¹²⁵ I-labeled pGEX-3X-MNprotein (2.27 μCi/μg of TCA-precipitable protein) was before use dilutedwith RIPA +1% FCS, and non-specifically binding radioactivity wasadsorbed with a suspension of fixed protein A-Staphylococcus aureuscells (SAC).

In an RIA for MN-specific antibodies, MAb-containing ascites fluids ortest sera were mixed with ¹²⁵ I-labeled protein and allowed to react ina total volume of 1 ml for 2 hours at room temperature. Subsequently, 50μl of a 10% suspension of SAC [Kessler, supra] was added and the mixturewas incubated for 30 minutes. Finally, the SAC was pelleted, 3× washedwith RIPA, and the bound radioactivity was determined on a gammacounter.

Titration of antibodies to MN antigen is shown in FIG. 11. Ascitic fluidfrom a mouse carrying M75 hybridoma cells (A) is shown to have a 50%end-point at dilution 1:1.4×10⁻⁶. At the same time, ascitic fluids withMAbs specific for MX protein (M16 and M67) showed no precipitation of¹²⁵ I-labeled pGEX-3X-MN even at dilution 1:200 (result not shown).Normal rabbit serum (C) did not significantly precipitate the MNantigen; rabbit anti-MaTu serum (B), obtained after immunization withlive MX-infected HeLa cells, precipitated 7% of radioactive MN protein,when diluted 1:200. The rabbit anti-MaTu serum is shown by immunoblot inExample 4 (above) to precipitate both MX and MN proteins.

Only one out of 180 human sera tested (90 control and 90 sera ofpatients with breast, ovarian or uterine cervical cancer) showed asignificant precipitation of the radioactively labeled MN recombinantprotein. That serum--L8--(D) was retested on immunoblot (as in Example4), but it did not precipitate any p54/58N from MX-infected HeLa cells.Also, six other human sera, including KH (E), were negative onimmunoblot. Thus, the only positive human serum in the RIA, L8, wasreactive only with the genetically engineered product, but not withnative p54/58N expressed by HeLa cells.

In an RIA for MN antigen, the dilution of MAb M75, which in the previoustest precipitated 50% of maximum precipitable radioactivity (=dilution1:1.4×10⁻⁶) was mixed with dilutions of cell extracts and allowed toreact for 2 hours. Then, ¹²⁵ I-labeled pGEX-3X-MN (25×10³ cpm/tube) wasadded for another 2 hours. Finally, the radioactivity bound to MAb M75was precipitated with SAC and washed as above. One hundred percentprecipitation (=0 inhibition) was considered the maximum radioactivitybound by the dilution of MAb used. The concentration of the MN antigenin the tested cell extracts was calculated from an inhibition curveobtained with "cold" pGEX-3X-MN, used as the standard (A in FIG. 12).

The reaction of radioactively labeled pGEX-3X-MN protein with MAb M75enabled us to quantitate MN antigen directly in cell extracts. FIG. 12shows that 3 ng of "cold" pGEX-3X-MN(A) caused a 50% inhibition ofprecipitation of "hot" pGEX-3X-MN; an equivalent amount of MN antigen ispresent in 3×10³ ng of proteins extracted from MaTu-infected HeLa (B) orfrom Rat2-Tk⁻ cells (C). Concentrations of MN protein in cell extracts,determined by this RIA, are presented in Table 1 below. It must beunderstood that the calculated values are not absolute, since MNantigens in cell extracts are of somewhat different sizes, and alsosince the genetically engineered MN protein is a product containingmolecules of varying size.

                  TABLE 1                                                         ______________________________________                                        Concentration of MN Protein in Cell Extracts                                  Cells        ng MN/mg total protein                                           ______________________________________                                        HeLa + MX    939.00                                                           Rat2-Tk.sup.-                                                                              1065.00                                                          HeLa         27.50                                                            XC           16.40                                                            T24           1.18                                                            HEF           0.00                                                            ______________________________________                                    

The data were calculated from the results shown in FIG. 12.

EXAMPLE 9 RIP of MX Antigen

An approximate concentration of p58X protein can be obtained by RIP fromextracts of MaTu-infected HeLa cells that have been metabolicallylabeled with [³⁵ S]-methionine or with a mixture of [¹⁴ C]-amino acids.The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Radioimmunoprecipitation of metabolically                                     labeled p58X protein                                                                       Radioactivity                                                                       Precipit. with                                                                          % cpm                                                               MAb M16 + SAC                                                                           in p58X                                          Inter-             Total     cpm × 10.sup.-3                                                                   (prec.                                 Label val    Cells     cpm × 10.sup.-6                                                                 Prec.1                                                                              Prec.2                                                                              1 + 2)                             ______________________________________                                        .sup.35 S                                                                           A      HeLa      7.850   11.455                                                                              7.631 0                                  Methi-       HeLa + MX 9.337   93.797                                                                              12.117                                                                              0.891                              onine B      HeLa      6.270    7.299                                                                              5.947 0                                               HeLa + MX 6.469   67.099                                                                              7.346 0.935                              .sup.14 C                                                                           A      HeLa      4.223    6.423                                                                              4.168 0                                  amino        HeLa + MX 3.577   29.280                                                                              4.936 0.705                              adids B      HeLa      3.266    4.915                                                                              3.805 0                                               HeLa + MX 2.627   24.323                                                                              4.346 0.824                              ______________________________________                                         Radioactivity counts are cpm of total or immunoprecipitated radioactivity     per dish. Intervals: A  cells labeled overnight; B  parallel cultures         after 24 hours' chase.                                                   

From the results shown in Table 2 it follows that p58X representsapproximately 0.8% of the proteins in the cell extracts.

Very similar values were obtained in cultures after overnight incubationwith labeled amino acids and in parallel cultures, which in additionwere incubated for another 24 hours in "cold" media with a fullcomplement of amino acids. Those results indicate that the values ofradioactivity obtained reflect already an equilibrium state, rather thanthe velocity of incorporation; therefore, the values cannot be verydifferent from the actual contents of p58X in the extracts. Extractsfrom cells labeled with [³⁵ S]-methionine gave values of p58X similar tothose of cells from extracts of cultures labeled with a mixture of [¹⁴C]-amino acids.

EXAMPLE 10 Immunoelectron and Scanning Microscopy of Control and ofMX-infected HeLa Cells

As indicated above in Example 1, MN antigen, detected by indirectimmunofluorescence with MAb M75, is located on the surface membranes andin the nuclei of MX-infected HeLa cells or in HeLa cells grown in densecultures. To elucidate more clearly the location of the MN antigen,immunoelectron microscopy was used wherein MAb M75 bound to MN antigenwas visualized with immunogold beads. [Herzog et al., "Colloidal goldlabeling for determining cell surface area," IN: Colloidal Gold, Vol. 3(Hayat, M. A., ed.), pp. 139-149 (Academic Press Inc.; San Diego,Calif.).]

Ultrathin sections of control and of MX-infected HeLa cells are shown inFIGS. 13A-13D. Those immuno-electron micrographs demonstrate thelocation of MN antigen in the cells, and in addition, the strikingultrastructural differences between control and MX-infected HeLa. Acontrol HeLa cell (FIG. 13A) is shown to have on its surface very littleMN antigen, as visualised with gold beads. The cell surface is rathersmooth, with only two little protrusions. No mitochondria can be seen inthe cytoplasm. In contrast, MX-infected HeLa cells (FIGS. 13B and C)show the formation of abundant, dense filamentous protrusions from theirsurfaces. Most of the MN antigen is located on those filaments, whichare decorated with immunogold. The cytoplasm of MX-infected HeLacontains numerous mitochondria (FIG. 13C). FIG. 13D demonstrates thelocation of MN antigen in the nucleus: some of the MN antigen is innucleoplasm (possibly linked to chromatin), but a higher concentrationof the MN antigen is in the nucleoli. Again, the surface of normal HeLa(panels A and E of FIG. 13) is rather smooth whereas MX-infected HeLacells have on their surface, numerous filaments and "blebs". Some of thefilaments appear to form bridges connecting them to adjacent cells.

It has been noted that in some instances of in vitro transformed cellscompared to their normal parent cells that one of the differences isthat the surface of normal cells was smooth whereas on the transformedcells were numerous hair-like protrusions [Darnell et al. "MolecularCell Biology," (2nd edition) Sci. Am. Books; W.H. Freeman and Co., NewYork (1990)]. Under that criteria MX-infected HeLa cells, as seen inFIG. 13F, has a supertransformed appearance.

Further in some tumors, amplification of mitochondria has been described[Bernhard, W., "Handbook of Molecular Cytology," pp. 687-715, Lima deFaria (ed.), North Holland Publishing Co.; Amsterdam-London (1972)].Such amplification was noted for MX-infected HeLa cells which stainedvery intensely with Janus' green, specific for mitochondria whereascontrol HeLa were only weakly stained.

It should be noted that electron microscopists were unable to find anystructural characteristics specific for tumor cells.

EXAMPLE 11 Antisense ODNs Inhibit MN Gene Expression

To determine whether both of the p54/58N proteins were encoded by onegene, the following experiments with antisense ODNs were performed.Previously sparse-growing HeLa cells were seeded to obtain anovercrowded culture and incubated for 130 hours either in the absence orin the presence of two gene-specific ODNs complementary to the 5' end ofMN mRNA. HeLa cells were subcultured at 8×10⁵ cells per ml of DMEM with10% FCS. Simultaneously, ODNs were added to the media as follows: (A)29-mer ODN1 (5' CGCCCAGTGGGTCATCTTCCCCAGAAGAG 3' [SEQ. ID. NO.: 3],complementary to positions 44-72) in 4 μM final concentration, (B)19-mer ODN2 (5' GGAATCCTCCTGCATCCGG 3' [SEQ. ID. NO.: 4], complementaryto positions 12-30) in 4 μM final concentration and (C) both ODN1 andODN2 in 2 μM final concentration each. (D) Cells treated in the sameway, but incubated without ODNs, served as a control. After 130 hours,extracts from the cells were prepared and analyzed by immunoblottingusing ¹²⁵ I-labeled MAb M75. Protein extracts from the cells wereanalyzed by immunoblotting and RIA using MAb M75. FIG. 3 provides theimmunoblot results of those experiments.

It was found that cultivation of HeLa cells with the ODNs resulted inconsiderable inhibition of p54/58N synthesis. The 19-mer ODN2 (FIG. 3B)in 4 μM final concentration was very effective; as determined by RIA, itcaused 40% inhibition, whereas the 29-mer ODN1 (4 μM) (FIG. 3A) and acombination of the two ODNs (FIG. 3C), each in 2 μM final concentration,were less effective in RIA showing a 25-35% increase of the MN-relatedproteins. At the same time, the amount of different HeLa cell proteindetermined by RIA using specific MAb H460 was in all cell variantsapproximately the same. Most importantly was that on immunoblot it couldbe seen that specific inhibition by the ODNs affected both of thep54/58N proteins. Thus, we concluded that the MN gene we cloned codedfor both p54/58N proteins in HeLa cells.

EXAMPLE 12 Northern Blotting of MN mRNA in Tumorigenic andNon-Tumorigenic Cell Lines

FIG. 4 shows the results of Northern blotting of MN mRNA in human celllines. Total RNA was prepared from the following cell lines by theguanidinium thiocyanate-CsCl method: HeLa cells growing in a dense (A)and sparse (B) culture; CGL1 (H/F-N) hybrid cells (C); CGL3 (D) and CGL4(E) segregants (both H/F-T); and human embryo fibroblasts (F). Fifteenμg of RNA were separated on a 1.2% formaldehyde gel and blotted onto aHybond C Super membrane (Amersham). MN cDNA NotI probe was labeled byrandom priming (Multiprime DNA labelling system; Amersham).Hybridization was carried out in the presence of 50% formamide at 42°C., and the final wash was in 0.1% SSPE and 0.1% SDS at 65° C. An RNAladder (0.24-9.5 kb) [Bethesda Research Laboratories (BRL); Bethesda,Md. (USA)] was used as a size standard. Membranes were exposed to filmsat -70° C., with intensifying screens.

Detected was a 1.5 kb MN-specific mRNA only in two tumorigenic segregantclones--CGL3 and CGL4 (H/F-T), but not in the non-tumorigenic hybridclone CGL1 (H/F-N) or in normal human fibroblasts. Further, the 1.5 kbmRNA was found in the HeLa cells growing in dense (FIG. 4A) but not insparse (FIG. 4B) culture.

Thus, the results of the Northern blotting were consistent with those ofthe above example in regard to MN-related proteins being associated withtumorigenicity.

EXAMPLE 13 Southern Blotting of Genomic DNAs from Different VertebrateSpecies to Detect MN Gene and Restriction Analysis of Genomic DNA ofHeLa Cells

FIG. 5 illustrates the detection of MN genes in the genomic DNAs ofvarious vertebrates by Southern blotting. Chromosomal DNA digested byPstI was as follows: (A) chicken; (B) bat; (C) rat; (D) mouse; (E)feline; (F) pig; (G) sheep; (H) bovine; (I) monkey; and (J) human HeLacells. Restriction fragments were separated on a 0.7% agarose gel andalkali blotted onto a Hybond N membrane (Amersham). The MN cDNA probelabelling and hybridization procedures were the same as for the Northernblotting analyses shown in FIG. 4 and described in Example 12. TheSouthern blot of FIG. 5 made with PstI indicates that the MN gene isconserved in all vertebrate genomes tested.

HeLa. Further, genomic DNA from HeLa cells was prepared as described byAusubel et al., Short Protocols in Molecular Biology [Greene PublishingAssociates and Wiley-Interscience; New York (1989)], digested withdifferent restriction enzymes, resolved on an agarose gel andtransferred to Hybond N+ membrane (Amersham). The HeLa genomic DNA wascleaved with the following restriction enzymes with the results shown inFIG. 17 (wherein the numbers in parentheses after the enzymes indicatethe respective lanes in FIG. 17): EcoRI (1), EcoRV (2), HindIII (3),KpnI (4), NcoI (5), PstI (6), and PvuII (7), and then analyzed bySouthern hybridization under stringent conditions using MN cDNA as aprobe.

The prehybridization and hybridization using an MN cDNA probe labelledwith ³² P-dCTP by random priming (Multiprime DNA labelling system;Amersham) as well as wash steps were carried out according to Amersham'sprotocols at high stringency. A 1 kb DNA Ladder [from Bethesda ResearchLaboratories (BRL); Bethesda, Md. (USA)] was used as a size standard.Membranes were exposed to films at -70° C., with intensifying screens.

The Southern blotting analysis of HeLa chromosomal DNA showed that thegene coding for MN is present in the human genome in a single copy (FIG.17). The sizes and distribution of MN-positive restriction fragmentsobtained using the restriction enzymes KpnI, NcoI and HindIII indicatethat the MN gene contains introns, since those enzymes cut the MNgenomic sequences despite the absence of their restriction sites in MNcDNA.

EXAMPLE 14 Immunohistochemical Staining of Tissue Specimens

To study and evaluate the tissue distribution range and expression of MNproteins, the monoclonal antibody M75 was used to stainimmunohistochemically a variety of human tissue specimens. The primaryantibody used in these immunohistochemical staining experiments was theM75 monoclonal antibody. A biotinylated second antibody andstreptavidin-peroxidase were used to detect the M75 reactivity insections of formalin-fixed, paraffin-embedded tissue samples. Acommercially available amplification kit, specifically the DAKO LSAB™kit [DAKO Corp., Carpinteria, Calif. (USA)] which provides matched,ready made blocking reagent, secondary antibody andsteptavidin-horseradish peroxidase was used in these experiments.

M75 immunoreactivity was tested according to the methods of thisinvention in multiple-tissue sections of breast, colon, cervical, lungand normal tissues. Such multiple-tissue sections were cut from paraffinblocks of tissues called "sausages" that were purchased from the City ofHope [Duarte, Calif. (USA)]. Combined in such a multiple-tissue sectionwere normal, benign and malignant specimens of a given tissue; forexample, about a score of tissue samples of breast cancers fromdifferent patients, a similar number of benign breast tissue samples,and normal breast tissue samples would be combined in one suchmultiple-breast-tissue section. The normal multiple-tissue sectionscontained only normal tissues from various organs, for example, liver,spleen, lung, kidney, adrenal gland, brain, prostate, pancreas, thyroid,ovary, and testis.

Also screened for MN gene expression were multiple individual specimensfrom cervical cancers, bladder cancers, renal cell cancers, and head andneck cancers. Such specimens were obtained from U.C. Davis MedicalCenter in Sacramento, Calif. and from Dr. Shu Y. Liao [Department ofPathology, St. Joseph Hospital, Orange, Calif.].

Controls used in these experiments were the cell lines CGL3 (H/F-Thybrid cells) and CGL1 (H/F-N hybrid cells) which are known to stainrespectively, positively and negatively with the M75 monoclonalantibody. The M75 monoclonal antibody was diluted to a 1:5000 dilutionwherein the diluent was either PBS [0.05 M phosphate buffered saline(0.15 M NaCl), pH 7.2-7.4] or PBS containing 1% protease-free BSA as aprotein stabilizer.

Immunohistochemical Staining Protocol

In brief, the sections were dewaxed, rehydrated and blocked to removenon-specific reactivity as well as endogenous peroxidase activity. Eachsection was then incubated with dilutions of the M75 monoclonalantibody. After the unbound M75 was removed by rinsing the section, thesection was sequentially reacted with a biotinylated antimouse IgGantibody and streptavidin conjugated to horseradish peroxidase; arinsing step was included between those two reactions and after thesecond reaction. Following the last rinse, the antibody-enzyme complexeswere detected by reaction with an insoluble chromogen (diaminobenzidine)and hydrogen peroxide. A positive result was indicated by the formationof an insoluble reddish-brown precipitate at the site of the primaryantibody reaction. The sections were then rinsed, counterstained withhematoxylin, dehydrated and cover slipped. Then the sections wereexamined using standard light microscopy. Following is the detailedimmunohistochemical staining protocol.

The paraffin-embedded, formalin-fixed tissue sections were mounted onglass microscopic slides. Each slide was labeled and placed in astaining rack. The tissues were deparaffinized by immersing the slidesin a series of 100%, 100%, 95%, 95% and 70% ethanol baths for 2minutes±1 minute each. The slides were then washed by immersing them intwo changes of deionized water for 2 minutes±1 minute each.

To block endogenous peroxidase activity, the slides were immersed in 3%hydrogen peroxide for 5 minutes±1 minute. The 3% hydrogen peroxide wasprepared by diluting 30% hydrogen peroxide 1:10 in deionized water.

A humidified chamber was prepared by placing an absorbent black clothmoistened with deionized water in a container with a lid. The chamberwas used to prevent evaporation of the reagents from the slides duringthe incubation steps which were all carried out at room temperature.

The slides were washed in 2 changes of PBS for 2 minutes±1 minute each.Excess liquid was drained from the slides, and any remaining liquid fromaround each tissue section was wiped away with a clean, lint-freeabsorbent tissue.

Each slide was then placed in the humidified chamber. A circle aroundeach tissue section was inscribed with a diamond-tip pen or ahydrophobic marker. Immediately after circling each section, theblocking reagent from the DAKO LSAB kit was applied in sufficientquantity (usually 100 μl) to cover the entire tissue section. The slideswere then incubated in the humidified chamber for at least 30 minutes.The slides were than drained, wiped and replaced in the chamber.

The diluted M75 antibody was then applied to each tissue section, andthe slides were incubated in the chamber for 60 minutes±5 minutes. Theslides were then drained, wiped and placed in a staining rack in a PBSbath (170-200 ml). The slides were then washed in 2 changes of PBS for 2minutes±1 minute each, drained and wiped to remove excess PBS andreplaced in the humidified chamber.

The secondary biotinylated antibody from the DAKO LSAB kit was appliedto each tissue section and incubated in the humidified chamber for 30minutes±2 minutes. The slides were then drained and wiped, and thenwashed in 2 changes of PBS for 2 minutes±1 minute each. Again the slideswere drained, wiped to remove excess PBS and replaced in the humidifiedchamber.

The streptavidin-peroxidase reagent was then applied to each tissuesection and incubated in the chamber for 30 minutes±2 minutes. Theslides were then drained, wiped and washed in 2 changes of PBS for 2minutes±1 minute each.

The chromogen-substrate solution was prepared by adding 90 μl of 30% H₂O₂ and 3 ml of diaminobenzidine [DAB Liquid Concentrate, Kirkegaard &Perry Laboratories (KPL), Inc.; Gaithersburg, Md. (USA)] to 150 ml ofTris buffer, and stirring to mix. The chromogen-substrate solution wasused within 30 minutes of mixing. The slides were dipped in thechromogen-substrate solution for 5-6 minutes, and then washed in 2changes of deionized water for 1-2 minutes each.

To elucidate the morphology of the tissue, the slides were then immersedin 1% Mayers modified hematoxylin counterstain [American HistologyReagent Company, Stockton, Calif. (USA)] for 2 minutes±1 minute. Theslides were then washed in several changes of tap water until the waterremained clear.

To intensify the hematoxylin stain, the slides were then immersed inblueing reagent (0.05% ammonium hydroxide) for 20 seconds±10 seconds.Then the slides were washed in 2 changes of deionized water for 3minutes ±1 minute total.

The tissue sections were then dehydrated by immersing the slides in aseries of 70%, 95%, 95%, 100% and 100% ethanol baths for 2 minutes±1minute each. The slides were then immersed in 3 separate changes ofxylene for 3 minutes±1 minute each. Upon removal of the slides from thelast xylene bath, 1-2 drops of mounting medium [Permount™; FisherScientific; Pittsburgh, Pa. (USA)] was applied to each stained tissuesection, and a coverslip was carefully laid over each section. After atleast 10 minutes, time allowing for the excess xylene to evaporate, theslides were viewed under a light microscope.

Interpretation. A deposit of a reddish brown precipitate over the plasmamembrane was taken as evidence that the M75 antibody had bound to a MNantigen in the tissue. The known positive control (CGL3) had to bestained to validate the assay. Section thickness was taken intoconsideration to compare staining intensities, as thicker sectionsproduce greater staining intensity independently of other assayparameters.

The above-described protocol was optimized for formalin-fixed tissues,but can be used to stain tissues prepared with other fixatives.

Results

Preliminary examination of cervical specimens showed that 62 of 68squamous cell carcinoma specimens (91.2%) stained positively with M75.Additionally, 2 of 6 adenocarcinomas and 2 of 2 adenosquamous cancers ofthe cervix also stained positively. In early studies, 55.6% (10 of 18)of cervical dysplasias stained positively. A total of 9 specimensincluding both cervical dysplasias and tumors, exhibited some MNexpression in normal appearing areas of the endocervical glandularepithelium, usually at the basal layer. In some specimens, whereasmorphologically normal-looking areas showed expression of MN antigen,areas exhibiting dysplasia and/or malignancy did not show MN expression.

M75 positive immunoreactivity was most often localized to the plasmamembrane of cells, with the most apparent stain being present at thejunctions between adjacent cells. Cytoplasmic staining was also evidentin some cells; however, plasma membrane staining was most often used asthe main criterion of positivity.

M75 positive cells tended to be near areas showing keratindifferentiation in cervical specimens. In some specimens, positivestaining cells were located in the center of nests of non-stainingcells. Often, there was very little, if any, obvious morphologicaldifference between staining cells and non-staining cells. In somespecimens, the positive staining cells were associated with adjacentareas of necrosis.

In most of the squamous cell carcinomas of the cervix, the M75immunoreactivity was focal in distribution, i.e., only certain areas ofthe specimen stained. Although the distribution of positive reactivitywithin a given specimen was rather sporadic, the intensity of thereactivity was usually very strong. In most of the adenocarcinomas ofthe cervix, the staining pattern was more homogeneous, with the majorityof the specimen staining positively.

Among the normal tissue samples, intense, positive and specific M75immunoreactivity was observed only in normal stomach tissues, withdiminishing reactivity in the small intestine, appendix and colon. Noother normal tissue stained extensively positively for M75.Occasionally, however, foci of intensely staining cells were observed innormal intestine samples (usually at the base of the crypts) or weresometimes seen in morphologically normal appearing areas of theepithelium of cervical specimens exhibiting dysplasia and/or malignancy.In such, normal appearing areas of cervical specimens, positive stainingwas seen in focal areas of the basal layer of the ectocervicalepithelium or in the basal layer of endocervical glandular epithelium.In one normal specimen of human skin, cytoplasmic MN staining wasobserved in the basal layer. The basal layers of these epithelia areusually areas of proliferation, suggesting the MN expression may beinvolved in cellular growth. In a few cervical biopsied specimens, MNpositivity was observed in the morphologically normal appearingstratified squamous epithelium, sometimes associated with cellsundergoing koilocytic changes.

Some colon adenomas (4 of 11) and adenocarcinomas (9 of 15) werepositively stained. One normal colon specimen was positive at the baseof the crypts. Of 15 colon cancer specimens, 4 adenocarcinomas and 5metastatic lesions were MN positive. Fewer malignant breast cancers (3of 25) and ovarian cancer specimens (3 of 15) were positively stained.Of 4 head and neck cancers, 3 stained very intensely with M75.

Although normal stomach tissue was routinely positive, 4 adenocarcinomasof the stomach were MN negative. Of 3 bladder cancer specimens (1adenocarcinoma, 1 non-papillary transitional cell carcinoma, and 1squamous cell carcinoma), only the squamous cell carcinoma was MNpositive. Approximately 40% (12 of 30) of lung cancer specimens werepositive; 2 of 4 undifferentiated carcinomas; 3 of 8 adenocarcinomas; 2of 8 oat cell carcinomas; and, 5 of 10 squamous cell carcinomas. Onehundred percent (4 of 4) of the renal cell carcinomas were MN positive.

In summary, MN antigen, as detected by M75 and immunohistochemistry inthe experiments described above, was shown to be prevalent in tumorcells, most notably in tissues of cervical cancers. MN antigen was alsofound in some cells of normal tissues, and sometimes in morphologicallynormal appearing areas of specimens exhibiting dysplasia and/ormalignancy. However, MN is not usually extensively expressed in mostnormal tissues, except for stomach tissues where it is extensivelyexpressed and in the tissues of the lower gastrointestinal tract whereit is less extensively expressed. MN expression is most often localizedto the cellular plasma membrane of tumor cells and may play a role inintercellular communication or cell adhesion. Representative results ofexperiments performed as described above are tabulated in Table 3.

                  TABLE 3                                                         ______________________________________                                        Immunoreactivity of M75 in Various Tissues                                                                POS/NEG                                           TISSUE           TYPE       (#pos/#tested)                                    ______________________________________                                        liver, spleen, lung,                                                                           normal     NEG (all)                                         kidney, adrenal gland,                                                        brain, prostate, pancreas,                                                    thyroid ovary, testis                                                         skin             normal     POS (in basal                                                                 layer) (1/1)                                      stomach          normal     POS                                               small intestine  normal     POS                                               colon            normal     POS                                               breast           normal     NEG (0/10)                                        cervix           normal     NEG (0/2)                                         breast           benign     NEG (0/17)                                        colon            benign     POS (4/11)                                        cervix           benign     POS (10/18)                                       breast           malignant  POS (3/25)                                        colon            malignant  POS (9/15)                                        ovarian          malignant  POS (3/15)                                        lung             malignant  POS (12/30)                                       bladder          malignant  POS (1/3)                                         head & neck      malignant  POS (3/4)                                         kidney           malignant  POS (4/4)                                         stomach          malignant  NEG (0/4)                                         cervix           malignant  POS (62/68)                                       ______________________________________                                    

The results recorded in this example indicate that the presence of MNproteins in a tissue sample from a patient may, in general, dependingupon the tissue involved, be a marker signaling that a pre-neoplastic orneoplastic process is occurring. Thus, one may conclude from theseresults that diagnostic/prognostic methods that detect MN antigen may beparticularly useful for screening patient samples for a number ofcancers which can thereby be detected at a pre-neoplastic stage or at anearly stage prior to obvious morphologic changes associated withdysplasia and/or malignancy being evident or being evident on awidespread basis.

EXAMPLE 15 Vaccine--Rat Model

As shown above in Example 7, in some rat tumors, for example, the XCtumor cell line (cells from a rat rhabdomyosarcoma), a rat MN protein,related to human MN, is expressed. Thus a model was afforded to studyantitumor immunity induced by experimental MN-based vaccines. Thefollowing representative experiments were performed.

Nine- to eleven-day-old Wistar rats from several families wererandomized, injected intraperitoneally with 0.1 ml of either control ratsera (the C group) or with rat serum against the MN fusion proteinpGEX-3X-MN (the IM group). Simultaneously both groups were injectedsubcutaneously with 10⁶ XC tumor cells.

Four weeks later, the rats were sacrificed, and their tumors weighed.The results are shown in FIG. 14. Each point on the graph represents atumor from one rat. The difference between the two groups--C and IM--wassignificant by Mann-Whitney rank test (U=84, α<0.025). The resultsindicate that the IM group of baby rats developed tumors about one-halfthe size of the controls, and 5 of the 18 passively immunized ratsdeveloped no tumor at all, compared to 1 of 18 controls.

The material listed below was deposited with the American Type CultureCollection (ATCC) in Manassas, Va. (USA). The deposits were made underthe provisions of the Budapest Treaty on the International Recognitionof Deposited Microorganisms for the Purposes of Patent Procedure andRegulations thereunder (Budapest Treaty). Maintenance of a viableculture is assured for thirty years from the date of deposit. Theorganism will be made available by the ATCC under the terms of theBudapest Treaty, and subject to an agreement between the Applicants andthe ATCC which assures unrestricted availability upon issuance of thepertinent U.S. Patent. Availability of the deposited strain is not to beconstrued as a license to practice the invention in contravention of therights granted under the authority of any Government in accordance withits patent laws.

    ______________________________________                                        Hybridoma     Deposit Date ATCC #                                             ______________________________________                                        VU-M75        September 17, 1992                                                                         HB 11128                                           ______________________________________                                    

The description of the foregoing embodiments of the invention have beenpresented for purposes of illustration and description. They are notintented to be exhaustive or to limit the invention to the precise formdisclosed, and obviously many modifications and variations are possiblein light of the above teachings. The embodiments were chosen anddescribed in order to explain the principles of the invention and itspractical application to enable thereby others skilled in the art toutilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated. It isintended that the scope of the invention be defined by the claimsappended hereto.

All references cited herein are hereby incorporated by reference.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS: 9                                               - <210> SEQ ID NO 1                                                           <211> LENGTH: 1399                                                            <212> TYPE: DNA                                                               <213> ORGANISM: HUMAN                                                         <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (1)..(1266)                                                   - <400> SEQUENCE: 1                                                           - cag agg ttg ccc cgg atg cag gag gat tcc cc - #c ttg gga gga ggc tct           48                                                                          Gln Arg Leu Pro Arg Met Gln Glu Asp Ser Pr - #o Leu Gly Gly Gly Ser           #                 15                                                          - tct ggg gaa gat gac cca ctg ggc gag gag ga - #t ctg ccc agt gaa gag           96                                                                          Ser Gly Glu Asp Asp Pro Leu Gly Glu Glu As - #p Leu Pro Ser Glu Glu           #             30                                                              - gat tca ccc aga gag gag gat cca ccc gga ga - #g gag gat cta cct gga          144                                                                          Asp Ser Pro Arg Glu Glu Asp Pro Pro Gly Gl - #u Glu Asp Leu Pro Gly           #         45                                                                  - gag gag gat cta cct gga gag gag gat cta cc - #t gaa gtt aag cct aaa          192                                                                          Glu Glu Asp Leu Pro Gly Glu Glu Asp Leu Pr - #o Glu Val Lys Pro Lys           #     60                                                                      - tca gaa gaa gag ggc tcc ctg aag tta gag ga - #t cta cct act gtt gag          240                                                                          Ser Glu Glu Glu Gly Ser Leu Lys Leu Glu As - #p Leu Pro Thr Val Glu           # 80                                                                          - gct cct gga gat cct caa gaa ccc cag aat aa - #t gcc cac agg gac aaa          288                                                                          Ala Pro Gly Asp Pro Gln Glu Pro Gln Asn As - #n Ala His Arg Asp Lys           #                 95                                                          - gaa ggg gat gac cag agt cat tgg cgc tat gg - #a ggc gac ccg ccc tgg          336                                                                          Glu Gly Asp Asp Gln Ser His Trp Arg Tyr Gl - #y Gly Asp Pro Pro Trp           #           110                                                               - ccc cgg gtg tcc cca gcc tgc gcg ggc cgc tt - #c cag tcc ccg gtg gat          384                                                                          Pro Arg Val Ser Pro Ala Cys Ala Gly Arg Ph - #e Gln Ser Pro Val Asp           #       125                                                                   - atc cgc ccc cag ctc gcc gcc ttc tgc ccg gc - #c ctg cgc ccc ctg gaa          432                                                                          Ile Arg Pro Gln Leu Ala Ala Phe Cys Pro Al - #a Leu Arg Pro Leu Glu           #   140                                                                       - ctc ctg ggc ttc cag ctc ccg ccg ctc cca ga - #a ctg cgc ctg cgc aac          480                                                                          Leu Leu Gly Phe Gln Leu Pro Pro Leu Pro Gl - #u Leu Arg Leu Arg Asn           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - aat ggc cac agt gtg caa ctg acc ctg cct cc - #t ggg cta gag atg gct          528                                                                          Asn Gly His Ser Val Gln Leu Thr Leu Pro Pr - #o Gly Leu Glu Met Ala           #               175                                                           - ctg ggt ccc ggg cgg gag tac cgg gct ctg ca - #g ctg cat ctg cac tgg          576                                                                          Leu Gly Pro Gly Arg Glu Tyr Arg Ala Leu Gl - #n Leu His Leu His Trp           #           190                                                               - ggg gct gca ggt cgt ccg ggc tcg gag cac ac - #t gtg gaa ggc cac cgt          624                                                                          Gly Ala Ala Gly Arg Pro Gly Ser Glu His Th - #r Val Glu Gly His Arg           #       205                                                                   - ttc cct gcc gag atc cac gtg gtt cac ctc ag - #c acc gcc ttt gcc aga          672                                                                          Phe Pro Ala Glu Ile His Val Val His Leu Se - #r Thr Ala Phe Ala Arg           #   220                                                                       - gtt gac gag gcc ttg ggg cgc ccg gga ggc ct - #g gcc gtg ttg gcc gcc          720                                                                          Val Asp Glu Ala Leu Gly Arg Pro Gly Gly Le - #u Ala Val Leu Ala Ala           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - ttt ctg gag gag ggc ccg gaa gaa aac agt gc - #c tat gag cag ttg ctg          768                                                                          Phe Leu Glu Glu Gly Pro Glu Glu Asn Ser Al - #a Tyr Glu Gln Leu Leu           #               255                                                           - tct cgc ttg gaa gaa atc gct gag gaa ggc tc - #a gag act cag gtc cca          816                                                                          Ser Arg Leu Glu Glu Ile Ala Glu Glu Gly Se - #r Glu Thr Gln Val Pro           #           270                                                               - gga ctg gac ata tct gca ctc ctg ccc tct ga - #c ttc agc cgc tac ttc          864                                                                          Gly Leu Asp Ile Ser Ala Leu Leu Pro Ser As - #p Phe Ser Arg Tyr Phe           #       285                                                                   - caa tat gag ggg tct ctg act aca ccg ccc tg - #t gcc cag ggt gtc atc          912                                                                          Gln Tyr Glu Gly Ser Leu Thr Thr Pro Pro Cy - #s Ala Gln Gly Val Ile           #   300                                                                       - tgg act gtg ttt aac cag aca gtg atg ctg ag - #t gct aag cag ctc cac          960                                                                          Trp Thr Val Phe Asn Gln Thr Val Met Leu Se - #r Ala Lys Gln Leu His           305                 3 - #10                 3 - #15                 3 -       #20                                                                           - acc ctc tct gac acc ctg tgg gga cct ggt ga - #c tct cgg cta cag ctg         1008                                                                          Thr Leu Ser Asp Thr Leu Trp Gly Pro Gly As - #p Ser Arg Leu Gln Leu           #               335                                                           - aac ttc cga gcg acg cag cct ttg aat ggg cg - #a gtg att gag gcc tcc         1056                                                                          Asn Phe Arg Ala Thr Gln Pro Leu Asn Gly Ar - #g Val Ile Glu Ala Ser           #           350                                                               - ttc cct gct gga gtg gac agc agt cct cgg gc - #t gct gag cca gtc cag         1104                                                                          Phe Pro Ala Gly Val Asp Ser Ser Pro Arg Al - #a Ala Glu Pro Val Gln           #       365                                                                   - ctg aat tcc tgc ctg gct gct ggt gac atc ct - #a gcc ctg gtt ttt ggc         1152                                                                          Leu Asn Ser Cys Leu Ala Ala Gly Asp Ile Le - #u Ala Leu Val Phe Gly           #   380                                                                       - ctc ctt ttt gct gtc acc agc gtc gcg ttc ct - #t gtg cag atg aga agg         1200                                                                          Leu Leu Phe Ala Val Thr Ser Val Ala Phe Le - #u Val Gln Met Arg Arg           385                 3 - #90                 3 - #95                 4 -       #00                                                                           - cag cac aga agg gga acc aaa ggg ggt gtg ag - #c tac cgc cca gca gag         1248                                                                          Gln His Arg Arg Gly Thr Lys Gly Gly Val Se - #r Tyr Arg Pro Ala Glu           #               415                                                           - gta gcc gag act gga gcc tagaggctgg atcttggaga at - #gtgagaag                1296                                                                          Val Ala Glu Thr Gly Ala                                                                   420                                                               - ccagccagag gcatctgagg gggagccggt aactgtcctg tcctgctcat ta - #tgccactt       1356                                                                          #                 139 - #9tttaaaat aaatatttat aat                             - <210> SEQ ID NO 2                                                           <211> LENGTH: 422                                                             <212> TYPE: PRT                                                               <213> ORGANISM: HUMAN                                                         - <400> SEQUENCE: 2                                                           - Gln Arg Leu Pro Arg Met Gln Glu Asp Ser Pr - #o Leu Gly Gly Gly Ser         #                 15                                                          - Ser Gly Glu Asp Asp Pro Leu Gly Glu Glu As - #p Leu Pro Ser Glu Glu         #             30                                                              - Asp Ser Pro Arg Glu Glu Asp Pro Pro Gly Gl - #u Glu Asp Leu Pro Gly         #         45                                                                  - Glu Glu Asp Leu Pro Gly Glu Glu Asp Leu Pr - #o Glu Val Lys Pro Lys         #     60                                                                      - Ser Glu Glu Glu Gly Ser Leu Lys Leu Glu As - #p Leu Pro Thr Val Glu         # 80                                                                          - Ala Pro Gly Asp Pro Gln Glu Pro Gln Asn As - #n Ala His Arg Asp Lys         #                 95                                                          - Glu Gly Asp Asp Gln Ser His Trp Arg Tyr Gl - #y Gly Asp Pro Pro Trp         #           110                                                               - Pro Arg Val Ser Pro Ala Cys Ala Gly Arg Ph - #e Gln Ser Pro Val Asp         #       125                                                                   - Ile Arg Pro Gln Leu Ala Ala Phe Cys Pro Al - #a Leu Arg Pro Leu Glu         #   140                                                                       - Leu Leu Gly Phe Gln Leu Pro Pro Leu Pro Gl - #u Leu Arg Leu Arg Asn         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Asn Gly His Ser Val Gln Leu Thr Leu Pro Pr - #o Gly Leu Glu Met Ala         #               175                                                           - Leu Gly Pro Gly Arg Glu Tyr Arg Ala Leu Gl - #n Leu His Leu His Trp         #           190                                                               - Gly Ala Ala Gly Arg Pro Gly Ser Glu His Th - #r Val Glu Gly His Arg         #       205                                                                   - Phe Pro Ala Glu Ile His Val Val His Leu Se - #r Thr Ala Phe Ala Arg         #   220                                                                       - Val Asp Glu Ala Leu Gly Arg Pro Gly Gly Le - #u Ala Val Leu Ala Ala         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Phe Leu Glu Glu Gly Pro Glu Glu Asn Ser Al - #a Tyr Glu Gln Leu Leu         #               255                                                           - Ser Arg Leu Glu Glu Ile Ala Glu Glu Gly Se - #r Glu Thr Gln Val Pro         #           270                                                               - Gly Leu Asp Ile Ser Ala Leu Leu Pro Ser As - #p Phe Ser Arg Tyr Phe         #       285                                                                   - Gln Tyr Glu Gly Ser Leu Thr Thr Pro Pro Cy - #s Ala Gln Gly Val Ile         #   300                                                                       - Trp Thr Val Phe Asn Gln Thr Val Met Leu Se - #r Ala Lys Gln Leu His         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Thr Leu Ser Asp Thr Leu Trp Gly Pro Gly As - #p Ser Arg Leu Gln Leu         #               335                                                           - Asn Phe Arg Ala Thr Gln Pro Leu Asn Gly Ar - #g Val Ile Glu Ala Ser         #           350                                                               - Phe Pro Ala Gly Val Asp Ser Ser Pro Arg Al - #a Ala Glu Pro Val Gln         #       365                                                                   - Leu Asn Ser Cys Leu Ala Ala Gly Asp Ile Le - #u Ala Leu Val Phe Gly         #   380                                                                       - Leu Leu Phe Ala Val Thr Ser Val Ala Phe Le - #u Val Gln Met Arg Arg         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Gln His Arg Arg Gly Thr Lys Gly Gly Val Se - #r Tyr Arg Pro Ala Glu         #               415                                                           - Val Ala Glu Thr Gly Ala                                                                 420                                                               - <210> SEQ ID NO 3                                                           <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: HUMAN                                                         - <400> SEQUENCE: 3                                                           #            29    ttcc ccagaagag                                             - <210> SEQ ID NO 4                                                           <211> LENGTH: 19                                                              <212> TYPE: DNA                                                               <213> ORGANISM: HUMAN                                                         - <400> SEQUENCE: 4                                                           # 19               cgg                                                        - <210> SEQ ID NO 5                                                           <211> LENGTH: 1522                                                            <212> TYPE: DNA                                                               <213> ORGANISM: HUMAN                                                         <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (13)..(1389)                                                  <220> FEATURE:                                                                <221> NAME/KEY: mat.sub.-- peptide                                            <222> LOCATION: (124)..(1389)                                                 - <400> SEQUENCE: 5                                                           - acagtcagcc gc atg gct ccc ctg tgc ccc agc ccc - # tgg ctc cct ctg ttg         51                                                                                        Met Ala Pr - #o Leu Cys Pro Ser Pro Trp Leu Pro Leu Leu         25                                                                            - atc ccg gcc cct gct cca ggc ctc act gtg ca - #a ctg ctg ctg tca ctg           99                                                                          Ile Pro Ala Pro Ala Pro Gly Leu Thr Val Gl - #n Leu Leu Leu Ser Leu           10                                                                            - ctg ctt ctg atg cct gtc cat ccc cag agg tt - #g ccc cgg atg cag gag          147                                                                          Leu Leu Leu Met Pro Val His Pro Gln Arg Le - #u Pro Arg Met Gln Glu           #          5                                                                  - gat tcc ccc ttg gga gga ggc tct tct ggg ga - #a gat gac cca ctg ggc          195                                                                          Asp Ser Pro Leu Gly Gly Gly Ser Ser Gly Gl - #u Asp Asp Pro Leu Gly           #     20                                                                      - gag gag gat ctg ccc agt gaa gag gat tca cc - #c aga gag gag gat cca          243                                                                          Glu Glu Asp Leu Pro Ser Glu Glu Asp Ser Pr - #o Arg Glu Glu Asp Pro           # 40                                                                          - ccc gga gag gag gat cta cct gga gag gag ga - #t cta cct gga gag gag          291                                                                          Pro Gly Glu Glu Asp Leu Pro Gly Glu Glu As - #p Leu Pro Gly Glu Glu           #                 55                                                          - gat cta cct gaa gtt aag cct aaa tca gaa ga - #a gag ggc tcc ctg aag          339                                                                          Asp Leu Pro Glu Val Lys Pro Lys Ser Glu Gl - #u Glu Gly Ser Leu Lys           #             70                                                              - tta gag gat cta cct act gtt gag gct cct gg - #a gat cct caa gaa ccc          387                                                                          Leu Glu Asp Leu Pro Thr Val Glu Ala Pro Gl - #y Asp Pro Gln Glu Pro           #         85                                                                  - cag aat aat gcc cac agg gac aaa gaa ggg ga - #t gac cag agt cat tgg          435                                                                          Gln Asn Asn Ala His Arg Asp Lys Glu Gly As - #p Asp Gln Ser His Trp           #    100                                                                      - cgc tat gga ggc gac ccg ccc tgg ccc cgg gt - #g tcc cca gcc tgc gcg          483                                                                          Arg Tyr Gly Gly Asp Pro Pro Trp Pro Arg Va - #l Ser Pro Ala Cys Ala           105                 1 - #10                 1 - #15                 1 -       #20                                                                           - ggc cgc ttc cag tcc ccg gtg gat atc cgc cc - #c cag ctc gcc gcc ttc          531                                                                          Gly Arg Phe Gln Ser Pro Val Asp Ile Arg Pr - #o Gln Leu Ala Ala Phe           #               135                                                           - tgc ccg gcc ctg cgc ccc ctg gaa ctc ctg gg - #c ttc cag ctc ccg ccg          579                                                                          Cys Pro Ala Leu Arg Pro Leu Glu Leu Leu Gl - #y Phe Gln Leu Pro Pro           #           150                                                               - ctc cca gaa ctg cgc ctg cgc aac aat ggc ca - #c agt gtg caa ctg acc          627                                                                          Leu Pro Glu Leu Arg Leu Arg Asn Asn Gly Hi - #s Ser Val Gln Leu Thr           #       165                                                                   - ctg cct cct ggg cta gag atg gct ctg ggt cc - #c ggg cgg gag tac cgg          675                                                                          Leu Pro Pro Gly Leu Glu Met Ala Leu Gly Pr - #o Gly Arg Glu Tyr Arg           #   180                                                                       - gct ctg cag ctg cat ctg cac tgg ggg gct gc - #a ggt cgt ccg ggc tcg          723                                                                          Ala Leu Gln Leu His Leu His Trp Gly Ala Al - #a Gly Arg Pro Gly Ser           185                 1 - #90                 1 - #95                 2 -       #00                                                                           - gag cac act gtg gaa ggc cac cgt ttc cct gc - #c gag atc cac gtg gtt          771                                                                          Glu His Thr Val Glu Gly His Arg Phe Pro Al - #a Glu Ile His Val Val           #               215                                                           - cac ctc agc acc gcc ttt gcc aga gtt gac ga - #g gcc ttg ggg cgc ccg          819                                                                          His Leu Ser Thr Ala Phe Ala Arg Val Asp Gl - #u Ala Leu Gly Arg Pro           #           230                                                               - gga ggc ctg gcc gtg ttg gcc gcc ttt ctg ga - #g gag ggc ccg gaa gaa          867                                                                          Gly Gly Leu Ala Val Leu Ala Ala Phe Leu Gl - #u Glu Gly Pro Glu Glu           #       245                                                                   - aac agt gcc tat gag cag ttg ctg tct cgc tt - #g gaa gaa atc gct gag          915                                                                          Asn Ser Ala Tyr Glu Gln Leu Leu Ser Arg Le - #u Glu Glu Ile Ala Glu           #   260                                                                       - gaa ggc tca gag act cag gtc cca gga ctg ga - #c ata tct gca ctc ctg          963                                                                          Glu Gly Ser Glu Thr Gln Val Pro Gly Leu As - #p Ile Ser Ala Leu Leu           265                 2 - #70                 2 - #75                 2 -       #80                                                                           - ccc tct gac ttc agc cgc tac ttc caa tat ga - #g ggg tct ctg act aca         1011                                                                          Pro Ser Asp Phe Ser Arg Tyr Phe Gln Tyr Gl - #u Gly Ser Leu Thr Thr           #               295                                                           - ccg ccc tgt gcc cag ggt gtc atc tgg act gt - #g ttt aac cag aca gtg         1059                                                                          Pro Pro Cys Ala Gln Gly Val Ile Trp Thr Va - #l Phe Asn Gln Thr Val           #           310                                                               - atg ctg agt gct aag cag ctc cac acc ctc tc - #t gac acc ctg tgg gga         1107                                                                          Met Leu Ser Ala Lys Gln Leu His Thr Leu Se - #r Asp Thr Leu Trp Gly           #       325                                                                   - cct ggt gac tct cgg cta cag ctg aac ttc cg - #a gcg acg cag cct ttg         1155                                                                          Pro Gly Asp Ser Arg Leu Gln Leu Asn Phe Ar - #g Ala Thr Gln Pro Leu           #   340                                                                       - aat ggg cga gtg att gag gcc tcc ttc cct gc - #t gga gtg gac agc agt         1203                                                                          Asn Gly Arg Val Ile Glu Ala Ser Phe Pro Al - #a Gly Val Asp Ser Ser           345                 3 - #50                 3 - #55                 3 -       #60                                                                           - cct cgg gct gct gag cca gtc cag ctg aat tc - #c tgc ctg gct gct ggt         1251                                                                          Pro Arg Ala Ala Glu Pro Val Gln Leu Asn Se - #r Cys Leu Ala Ala Gly           #               375                                                           - gac atc cta gcc ctg gtt ttt ggc ctc ctt tt - #t gct gtc acc agc gtc         1299                                                                          Asp Ile Leu Ala Leu Val Phe Gly Leu Leu Ph - #e Ala Val Thr Ser Val           #           390                                                               - gcg ttc ctt gtg cag atg aga agg cag cac ag - #a agg gga acc aaa ggg         1347                                                                          Ala Phe Leu Val Gln Met Arg Arg Gln His Ar - #g Arg Gly Thr Lys Gly           #       405                                                                   - ggt gtg agc tac cgc cca gca gag gta gcc ga - #g act gga gcc                 #1389                                                                         Gly Val Ser Tyr Arg Pro Ala Glu Val Ala Gl - #u Thr Gly Ala                   #   420                                                                       - tagaggctgg atcttggaga atgtgagaag ccagccagag gcatctgagg gg - #gagccggt       1449                                                                          - aactgtcctg tcctgctcat tatgccactt ccttttaact gccaagaaat tt - #tttaaaat       1509                                                                          #    1522                                                                     - <210> SEQ ID NO 6                                                           <211> LENGTH: 459                                                             <212> TYPE: PRT                                                               <213> ORGANISM: HUMAN                                                         - <400> SEQUENCE: 6                                                           - Met Ala Pro Leu Cys Pro Ser Pro Trp Leu Pr - #o Leu Leu Ile Pro Ala         25                                                                            - Pro Ala Pro Gly Leu Thr Val Gln Leu Leu Le - #u Ser Leu Leu Leu Leu         10                                                                            - Met Pro Val His Pro Gln Arg Leu Pro Arg Me - #t Gln Glu Asp Ser Pro         #                10                                                           - Leu Gly Gly Gly Ser Ser Gly Glu Asp Asp Pr - #o Leu Gly Glu Glu Asp         #             25                                                              - Leu Pro Ser Glu Glu Asp Ser Pro Arg Glu Gl - #u Asp Pro Pro Gly Glu         #         40                                                                  - Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro Gl - #y Glu Glu Asp Leu Pro         #     55                                                                      - Glu Val Lys Pro Lys Ser Glu Glu Glu Gly Se - #r Leu Lys Leu Glu Asp         # 75                                                                          - Leu Pro Thr Val Glu Ala Pro Gly Asp Pro Gl - #n Glu Pro Gln Asn Asn         #                 90                                                          - Ala His Arg Asp Lys Glu Gly Asp Asp Gln Se - #r His Trp Arg Tyr Gly         #            105                                                              - Gly Asp Pro Pro Trp Pro Arg Val Ser Pro Al - #a Cys Ala Gly Arg Phe         #       120                                                                   - Gln Ser Pro Val Asp Ile Arg Pro Gln Leu Al - #a Ala Phe Cys Pro Ala         #   135                                                                       - Leu Arg Pro Leu Glu Leu Leu Gly Phe Gln Le - #u Pro Pro Leu Pro Glu         140                 1 - #45                 1 - #50                 1 -       #55                                                                           - Leu Arg Leu Arg Asn Asn Gly His Ser Val Gl - #n Leu Thr Leu Pro Pro         #               170                                                           - Gly Leu Glu Met Ala Leu Gly Pro Gly Arg Gl - #u Tyr Arg Ala Leu Gln         #           185                                                               - Leu His Leu His Trp Gly Ala Ala Gly Arg Pr - #o Gly Ser Glu His Thr         #       200                                                                   - Val Glu Gly His Arg Phe Pro Ala Glu Ile Hi - #s Val Val His Leu Ser         #   215                                                                       - Thr Ala Phe Ala Arg Val Asp Glu Ala Leu Gl - #y Arg Pro Gly Gly Leu         220                 2 - #25                 2 - #30                 2 -       #35                                                                           - Ala Val Leu Ala Ala Phe Leu Glu Glu Gly Pr - #o Glu Glu Asn Ser Ala         #               250                                                           - Tyr Glu Gln Leu Leu Ser Arg Leu Glu Glu Il - #e Ala Glu Glu Gly Ser         #           265                                                               - Glu Thr Gln Val Pro Gly Leu Asp Ile Ser Al - #a Leu Leu Pro Ser Asp         #       280                                                                   - Phe Ser Arg Tyr Phe Gln Tyr Glu Gly Ser Le - #u Thr Thr Pro Pro Cys         #   295                                                                       - Ala Gln Gly Val Ile Trp Thr Val Phe Asn Gl - #n Thr Val Met Leu Ser         300                 3 - #05                 3 - #10                 3 -       #15                                                                           - Ala Lys Gln Leu His Thr Leu Ser Asp Thr Le - #u Trp Gly Pro Gly Asp         #               330                                                           - Ser Arg Leu Gln Leu Asn Phe Arg Ala Thr Gl - #n Pro Leu Asn Gly Arg         #           345                                                               - Val Ile Glu Ala Ser Phe Pro Ala Gly Val As - #p Ser Ser Pro Arg Ala         #       360                                                                   - Ala Glu Pro Val Gln Leu Asn Ser Cys Leu Al - #a Ala Gly Asp Ile Leu         #   375                                                                       - Ala Leu Val Phe Gly Leu Leu Phe Ala Val Th - #r Ser Val Ala Phe Leu         380                 3 - #85                 3 - #90                 3 -       #95                                                                           - Val Gln Met Arg Arg Gln His Arg Arg Gly Th - #r Lys Gly Gly Val Ser         #               410                                                           - Tyr Arg Pro Ala Glu Val Ala Glu Thr Gly Al - #a                             #           420                                                               - <210> SEQ ID NO 7                                                           <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: HUMAN                                                         - <400> SEQUENCE: 7                                                           #               25 ctcc aggag                                                 - <210> SEQ ID NO 8                                                           <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: HUMAN                                                         - <400> SEQUENCE: 8                                                           #              26  ccct cttctt                                                - <210> SEQ ID NO 9                                                           <211> LENGTH: 48                                                              <212> TYPE: DNA                                                               <213> ORGANISM: HUMAN                                                         <220> FEATURE:                                                                <221> NAME/KEY: primer.sub.-- bind                                            <222> LOCATION: (1)..(48)                                                     - <400> SEQUENCE: 9                                                           #                48acgc gtcgactagt acgggnnggg nngggnng                        __________________________________________________________________________

What we claim is:
 1. A method of delivering a chemotherapeutic agent ortoxic agent to a vertebrate cancer cell, which is abnormally expressingMN protein, wherein said method comprises contacting the cell with anantibody, or a biologically active antibody fragment, which antibody orantibody fragment specifically binds to a cell surface epitope of an MNprotein and is linked to a chemotherapeutic agent or toxic agent;whereinsaid MN protein is encoded by a nucleotide sequence selected from thegroup consisting of: (a) SEQ ID NOS: 1 and 5; (b) nucleotide sequencesthat hybridize under stringent hybridization conditions of 0.15 to 0.9 Msalt in the presence of 50% formamide at 42° C. to the complements ofSEQ ID NOS: 1 and 5, with a final wash of 0.1% SSPE and 0.1% SDS at 65°C.; and (c) nucleotide sequences that differ from SEQ ID NOS: 1 and 5,or from the nucleotide sequences of (b) in codon sequence due to thedegeneracy of the genetic code; wherein said cancer cell is not fromstomach tissue.
 2. A method of treating a pre-neoplastic or neoplasticdisease characterized by abnormal expression of MN protein in a patientcomprising administering to said patient a therapeutically effectiveamount of a composition comprising an antibody or a biologically activeantibody fragment which specifically binds to a cell surface epitope ofan MN protein; wherein said MN protein is encoded by a nucleotidesequence selected from the group consisting of:(a) SEQ ID NOS: 1 and 5;(b) nucleotide sequences that hybridize under stringent hybridizationconditions of 0.15 to 0.9 M salt in the presence of 50% formamide at 42°C. to the complements of SEQ ID NOS: 1 and 5, with a final wash of 0.1%SSPE and 0.1% SDS at 65° C.; and (c) nucleotide sequences that differfrom SEQ ID NOS: 1 and 5, or from the nucleotide sequences of (b) incodon sequence due to the degeneracy of the genetic code; wherein saidpre-neoplastic or neoplastic disease is not of said patient's stomach.3. The method according to claim 1 wherein said toxic agent is ricin A.4. The method according to claim 1 wherein said antibody is a monoclonalantibody.
 5. The method according to claim 1 wherein said cancer cell iscontacted with said biologically active antibody fragment linked to achemotherapeutic or toxic agent.
 6. The method according to claim 4wherein said monoclonal antibody is designated M75 which is secretedfrom hybridoma HB 11128, deposited at the American Type CultureCollection (ATCC).
 7. The method according to claim 1 wherein saidantibody was prepared against MaTu-infected HeLa cells.
 8. The methodaccording to claim 5 wherein said biologically active antibody fragmentwas genetically engineered.
 9. The method according to claim 4 whereinsaid monoclonal antibody is humanized.
 10. The method according to claim1 wherein said antibody was prepared against a recombinantly produced MNprotein, MN fusion protein or MN polypeptide.
 11. The method accordingto claim 10 wherein said MN fusion protein consists of MN protein andglutathione S-transferase.
 12. The method according to claim 1 whereinsaid antibody was prepared against an MN polypeptide.
 13. The methodaccording to claim 2 wherein said antibody was prepared against an MNprotein, an MN fusion protein or an MN polypeptide, wherein said MNprotein, MN fusion protein and MN polypeptide were recombinantlyproduced.
 14. The method according to claim 13 wherein said MN fusionprotein consists of MN protein and glutathione S-transferase.
 15. Themethod according to claim 2 wherein said antibody was prepared againstMaTu-infected HeLa cells.
 16. The method according to claim 2 whereinsaid antibody was prepared against an MN polypeptide.
 17. The methodaccording to claim 2 wherein said antibody is a monoclonal antibody. 18.The method according to claim 17 wherein said antibody is designated M75and is secreted from the hybridoma HB 11128, deposited at the AmericanType Culture Collection (ATCC).
 19. The method according to claim 2wherein said patient is administered a therapeutically effective amountof a composition comprising said biologically active antibody fragment.20. The method according to claim 19 wherein said antibody fragment isgenetically engineered.
 21. The method according to claim 17 whereinsaid monoclonal antibody is humanized.
 22. A method of inhibiting growthof a vertebrate pre-neoplastic or neoplastic cell, which is abnormallyexpressing MN protein, comprising contacting that cell with an effectivegrowth-inhibiting amount of a composition comprising an antibody or abiologically active antibody fragment which specifically binds to an MNantigen, wherein said antibody or said biologically active antibodyfragment is linked to a chemotherapeutic agent or toxic agent; whereinsaid MN antigen is encoded by a nucleotide sequence selected from thegroup consisting of:(a) SEQ ID NO: 5; (b) nucleotide sequences thathybridize under stringent hybridization conditions of 0.1 5 to 0.9 Msalt in the presence of 50% formamide at 42° C. to the complement of SEQID NO: 5, with a final wash of 0.1% SSPE and 0.1% SDS at 65° C.; and (c)nucleotide sequences that differ from SEQ ID NO: 5, or from thenucleotide sequences of (b) in codon sequence due to the degeneracy ofthe genetic code; and wherein said cell is not from a stomach tissue.23. The method according to claim 22 wherein said pre-neoplastic orneoplastic cell is a mammalian cell.
 24. The method according to claim22 wherein said pre-neoplastic or neoplastic cell is a human cell. 25.The method according to claim 22 wherein said antibody or biologicallyactive antibody fragment is monoclonal.
 26. The method according toclaim 25 wherein said antibody is designated M75 and is secreted fromhybridoma HB 11128, deposited at the American Type Culture Collection(ATCC).
 27. The method according to claim 22 wherein an amount of saidcomposition comprising said antibody or biologically active antibodyfragment, that is linked to a chemotherapeutic or toxic agent, isadministered to a human patient having cancer, wherein said amount iseffective to inhibit said cancer's growth.